Safety of Continuously Infused High Dose Vitamin C

April 19, 2012

This study was done in conjunction with a series of studies by the Matsuda group that involved the use of high doses of infused vitamin C in severely burned subjects.  They had been testing high dose vitamin C as an anti-oxidant in an attempt to diminish the delayed injury that always occurred in severely burned patients. Their initial studies involved rats, guinea pigs and eventually anesthetized dogs to show the efficacy of this technique.  There was some resistance in the medical community to using these rather high doses of vitamin C, especially in patients with such a disturbed fluid and salt balance as those with severe or extensive burns.

This study was done to show that high dose vitamin C was not harmful in normal human subjects with the implication being that it may also be safe in burned subjects.  Interestingly, this therapy has, in the years since performing this study, become a fairly widely used modality for extensively burned subjects and has proven to be very effective in stabilizing the otherwise precariously unbalanced hemodynamic condition of these patients.

The full paper and a link to the PDF are shown below.

Safety of Continuously Infused High dose Vitamin C

The Safety of Continuous Intravenous Infusion Of

High‑dose Vitamin C in Healthy Humans

by

Takayoshi Matsuda, M.D., Hideki Yuasa, M.D., Walid Khabaz, M.D.,

Candice Richardson, Pharm D., Marella Hanumadass, M.D.,

and Robert J. Walter, Ph.D.

Burn Center and Department of Surgery, Cook County Hospital;

Department of Surgery, University of Illinois;

Hektoen Institute for Medical Research, Chicago, IL

Presented at the Twenty‑sixth Annual Meeting of the American

Burn Association, Orlando, Florida, April 20‑23, 1994.

Address all correspondence to:

            Robert J. Walter, Ph D.

Division of Surgical Research

Department of Surgery

Cook County Hospital

627 South Wood Street

Chicago, IL  60612

Tel:  (312)-633-7237 (office)

Fax:  (312)-633-8347

ABSTRACT

Background:  It has been shown that continuous intravenous infusion of vitamin C in high doses (14 mg/kg/hr) decreases postburn capillary permeability and reduces the resuscitation fluid volume requirements by 75% in burned animals. 

Methods:  In the present study, the effects of an 8 hr intravenous infusion of high‑dose vitamin C (0.5, 1.0, and 2.0 gm/hr) were evaluated in 4 healthy adult volunteers. 

Results:  Plasma vitamin C levels ranged from 4 to 16 μg/ml at baseline, and increased to 41-90 μg/ml during the infusion of 0.5 g/hr vitamin C, to 92-152 μg/ml during the 1 g/hr infusion, and to 162-290 μg/ml during the 2 g/ hr infusion.  Plasma vitamin C values decreased rapidly after termination of the infusion and returned to baseline by 16 hours postinfusion.  None of the subjects developed any subjective symptoms.  Vital signs, EKG monitoring, urinalysis, and hematology and chemistry blood tests before, during, and after the vitamin C infusion were all within normal limits. 

Conclusions:  Continuous intravenous infusion of up to 2 g/hr of vitamin C for 8 hrs appeared to have no adverse effects on healthy adult humans.

Key Words: VITAMIN C,  SAFETY,  ANTIOXIDANT,  HIGH‑DOSE

INTRODUCTION

Burn injuries cause increased capillary permeability which results in extensive fluid and protein leakage from the intravascular space.  For extensively burned patients, a massive volume of fluid is required for resuscitation during the first 24 hours postburn in order to prevent hypovolemic shock.  Friedl and associates have demonstrated that increased postburn capillary permeability is due to capillary endothelial damage caused by free radicals generated by the increased activity of xanthine oxidase ¹.  We have previously demonstrated in animal studies that continuous intravenous infusion of a natural antioxidant, vitamin C, in high doses (14 mg/kg/hr) minimizes the burn tissue lipid peroxidation ² and reduces fluid and protein leakage from the intravascular space to the interstitial space ³.  We have also shown that, for guinea pigs with 70% body surface area (BSA) burns, the resuscitation fluid volume requirements can be reduced by 75% with adjuvant administration of vitamin C ⁴.  It has also been reported that the administration of vitamin C must be continued for at least 8 hours in order to maintain adequate hemodynamic stability when the burned animals are resuscitated with a reduced fluid volume ⁵.

Before this high‑dose vitamin C therapy can be advanced to a clinical trial in burned patients, the possible toxicity or adverse side effects of such therapy must be determined.  The present study was undertaken to evaluate the safety of short‑term intravenous infusion of high‑dose vitamin C in healthy human volunteers.

SUBJECTS AND METHODS

Study subjects.  Four (4) healthy male volunteers, ages 28 to 34 years with body weights ranging from 68 to 90 kg, participated in the study.  The experimental protocol was approved by the Institutional Review Board of the Cook County Hospital and each subject signed an informed consent.  There was no financial payment to the volunteers for their participation in the study.  One week prior to the initiation of the study, a complete history was obtained, a physical examination performed, and baseline blood samples drawn.  The history, the physical examination, and all the blood test values were confirmed to be within normal limits before the initiation of the study.  None of the subjects had a history of kidney disease, and none were taking routine vitamin C supplements.

Preparation of Vitamin C Solution.  An injectable vitamin C solution, Cenolate (500 mg/ml; Abbott Laboratories Hospital Products Division, Abbott Park, IL), was diluted with sterile water at a ratio of 1 to 17.44 (vol/vol).  The concentration of vitamin C and sodium in this diluted sodium ascorbate solution was 27.11 mg/ml and 154 mEq/L, respectively.

Dosing Schedule.  Three different doses of vitamin C (i.e., 8 hour infusion of 0.5, 1.0 and 2.0 g/hr) were studied in each of the subjects with one week intervals between each infusion.  In the first week, a loading dose of 0.5 g vitamin C was infused over 20 minutes, followed by a continuous infusion of 0.5 g/hr of vitamin C (0.26 ml/kg/hr) for 8 hours.  In the second and third weeks, 1.0 g/hr and 2.0 g/hr doses of vitamin C were studied, respectively, using an identical scheme.

Conduct of the study.  The study was performed on the Intensive Care Unit of the Burn Center at Cook County Hospital.  On the morning of the study day, the subject was assigned to a bed with ambulation privileges ad libitum.  The subject remained in the Burn Center for approximately 24 hours and then returned to the Burn Center as required for follow‑up data collection.  A regular hospital diet was provided and water was given ad libitum throughout the study period.

A catheter was inserted into a forearm vein for blood drawing and then kept open with a heparin lock for subsequent blood samplings at predetermined intervals during and after the infusion of vitamin C.  After obtaining a blood sample for the baseline measurement, a venous catheter was inserted into the opposite forearm and the vitamin C infusion was initiated.

           

            Monitoring and Data Collection.  Vital signs were measured every hour during the infusion, and every 4 hours thereafter until 12 hours postinfusion.  The subject was placed on a continuous EKG monitor.  Fluid intake and urine output during the study day were recorded.  Urinalysis (specific gravity, pH, sugar, acetone, protein, blood, and microscopic examination) was performed prior to the vitamin C infusion, during the 24‑hour study period (i.e., 8 hours infusion and 16 hours postinfusion), and at 24 hours and 7 days postinfusion.  Hematological tests (CBC and coagulation profile) and blood chemistries (sodium, potassium, chloride, bicarbonate, calcium, glucose, total protein, albumin, globulin, phosphorus, cholesterol, urea nitrogen, creatinine, alkaline phosphatase, SGOT, SGPT, GGT, and LDH) were performed at baseline, at the end of the vitamin C administration, and at 1 and 7 days postinfusion.  Tests for urine and blood as described above were determined in the hospital's central clinical laboratories.

Blood samples for venous blood gases were drawn at baseline, and at 1, 2, 4 and 8 hours during the vitamin C infusion.  These results were determined in the Neonatology Stat Laboratory of Cook County Hospital.  Plasma vitamin C levels were drawn at baseline, and at 1, 2, 4, and 8 hours during the vitamin C infusion, and analyzed in the Burn Center research laboratory using HPLC ⁶.

RESULTS

Each of the four subjects completed the three‑week study for the evaluation of the three different doses of vitamin C.  None of the subjects developed any subjective symptoms that may be related to drug reaction such as headache, dizziness, nausea, vomiting, abdominal pain, or diarrhea.  Vital signs monitored during the study periods remained essentially within normal limits. There were no abnormalities detected in the EKG monitoring.  All the laboratory measurements before, during, and after the vitamin C administration in each of the subjects were within normal limits.  Follow‑up examinations and laboratory tests of all the subjects both one week and one month after the 2 g/hr infusion revealed no abnormalities.

The plasma vitamin C levels of one of the subjects before, during, and after the infusion of the three different doses of vitamin C are shown in Figure 1.  The plasma vitamin C levels of all four subjects at 2 g/hr dose are illustrated in Figure 2.  Plasma vitamin C levels of all the subjects ranged from 4-16 μg/ml prior to the vitamin C infusion.  Levels increased to 41-90 μg/ml during the infusion of 0.5 g/hr vitamin C, to 92-152 μg/ml during the 1 g/hr infusion, and to 162-290 μg/ml during the 2 g/hr infusion.  The plasma vitamin C values of all the subjects decreased rapidly after termination of the infusion, and returned to baseline by 16 hours postinfusion.

DISCUSSION

Vitamin C (ascorbic acid) is a six-carbon, water‑soluble vitamin which is structurally related to glucose and other hexoses ⁷.  Its physiological functions are numerous.  Severe or prolonged deficiency leads to the clinical condition known as scurvy.  The recommended daily dietary allowance of vitamin C for the healthy adult is 60 mg/day ⁸, which will prevent signs of scurvy for at least 4 weeks.  Vitamin C is also a well-known naturally-occurring antioxidant ^9‑11.  We have previously shown that continuous intravenous infusion of vitamin C in high doses (14 mg/kg/hr) decreases burn tissue lipid peroxidation ², minimizes postburn capillary permeability ³, and reduces the resuscitation fluid volume requirements in burned animals by 75% ⁴.  When vitamin C is administered via intravenous infusion, it is impossible to elevate the concentration of vitamin C selectively in the burned area only, instead the concentration of vitamin C in the total extracellular fluid must be elevated to the level required by the burned tissue.  This resulting plasma concentration of vitamin C is dependent upon the dose of vitamin C administered.  The minimum dose of vitamin C that was necessary to achieve the desired therapeutic effects in our animal experiments was 14 mg/kg/hr ⁴.  This dosage, equivalent to 1,000 mg/hr for a 70 kg adult, is very large as compared to the physiological maintenance dose (60 mg/day) and represents a pharmacological dose.

Pauling ¹² has advocated the ingestion of large doses of vitamin C as an antioxidant for health maintenance.  His recommended daily dose is 0.25 to 5 grams for a healthy adult based upon the extrapolation of the rate of vitamin C synthesis observed in animals ¹².  In addition, he recommends an even greater dosage, 5 to 20 g/day, for the treatment of illnesses such as the common cold ¹³.  The efficacy and safety of so‑called "megadose" vitamin C ingestion for the common cold, however, is controversial.  

Rivers, one of the organizers of the "Third New York Academy of Sciences Conference on Vitamin C" held in 1986, concluded ¹⁴ that the use of large quantities of ascorbic acid did not result in the production of calcium‑oxalate stones, increased uric acid excretion, impaired vitamin B12 status, iron overload, systemic conditioning, or increased mutagenic activity in healthy individuals.  The only contraindication regarding the ingestion of large quantities of vitamin C mentioned in that paper ¹⁴ relates to patients with renal impairment and patients on chronic hemodialysis.  Furthermore, any reported criticism of "megadose" vitamin C ingestion has been directed toward chronic or long‑term usage of high-dose vitamin C.  To our knowledge, the safety of short‑term (e.g., 8 to 24 hours) intravenous infusion of high‑dose, neutral pH, vitamin C has not been evaluated elsewhere.  

In the present study, none of the subjects developed any overt symptoms nor abnormal laboratory values associated with the high‑dose vitamin C infusion.  Although the number of subjects studied was small, it appears that continuous intravenous infusion of up to 2 grams per hour of vitamin C for 8 hours is not detrimental to healthy adult humans.

Vitamin C (ascorbic acid) has a low molecular weight (176 g/mol), and passes freely through the glomerular filtration barriers.  Reabsorption of ascorbate from glomerular filtrate in the renal tubules is an active, saturable process ¹⁴.  It has been shown that the reabsorption mechanism is saturated at plasma levels of 8 to 9 μg/ml ^15,16.  Plasma ascorbate in excess of that required to maintain plasma levels at approximately 10 μg/ml is, therefore, efficiently eliminated by the kidney.  The rapid decrease in the plasma ascorbate level after the termination of vitamin C infusion in the present study supports this contention.  The continuous high plasma ascorbate levels depicted during the infusion of vitamin C in the present study suggests that intake has exceeded the elimination capability of the kidneys resulting in elevation of plasma ascorbate levels.

REFERENCES

1.  Freidl HP, Till GO, Trentz O:  Roles of histamine, complement and xanthine oxidase in thermal injury of skin. Amer J Pathol 135:203, 1989.

2.  Matsuda T, Tanaka H, Yuasa H:  The effects of high‑dose vitamin C therapy on postburn lipid peroxidation. J Burn Care Rehabil 14:624, 1993.

3.  Matsuda T, Tanaka H, Hanumadass M:  Effects of high‑dose vitamin C administration on postburn microvascular fluid and protein flux. J Burn Care Rehabil 13:560, 1992.

4.  Matsuda T, Tanaka H, Williams S:  Reduced fluid volume requirement for resuscitation of third degree burns using high dose vitamin C. J Burn Care Rehabil 12:525, 1991.

5.  Tanaka H, Broaderick P, Shimazaki S:  How long do we need to give antioxidant therapy during resuscitation when its administration is delayed for two hours? J Burn Care Rehabil 13:567, 1992.

6.  Dennison DB, Brawley TG, Hunter GL:  Rapid high‑performance liquid chromatographic determination of ascorbic acid and combined ascorbic acid‑dehydroascorbic acid in beverages. J Agr Food Chem 29:927, 1981.

7.  Gilman A, Goodman L, Rall T., eds. The phamacological basis of therapeutics. New York: MacMillan Publishing Co. 1985; 1567‑1568.

8.  Food and Nutrition Board, National Research Council. Recommended dietary allowances. 1989; 10th edition:p 118 Washington, DC. National Academy of Sciences.

9.  Nishikimi M:  Oxidation of ascorbic acid with superoxide anion generated by the xanthine‑xanthine oxidase system. Biochem Biophys Res Comm 63:463, 1975.

10.  Bielski BHJ, Richter HW, Chan PC:  Some properties of the ascorbate free radical. Ann NY Acad Sci 258:231, 1975.

11.  Bodannes RS, Chan PC:  Ascorbic acid as a scavenger of singlet oxygen. FEBS Letters 105:195, 1979.

12.  Pauling L. Vitamin C, the common cold, and the flu. San Francisco: W.H.Freeman and Company, 1976; 145‑146.

13.  Pauling L. Vitamin C, the common cold, and the flu. San Francisco: W.H.Freeman and Company, 1976; 148

14.  Rivers JM:  Safety of high‑level vitamin C ingestion. Ann NY Acad Sci 498:445, 1987.

15.  Hagler L, Herman RH:  Oxalate metabolism.  I. Amer J Clin Nutr 26:758, 1973.

16.  Hagler L, Herman RH:  Oxalate metabolism.  II. Amer J Clin Nutr 26:882, 1973.

FIGURES

 Figure 1.  The plasma vitamin C levels of one of the subjects before, during, and after the infusion of three different doses of vitamin C (i.e., 0.5, 1.0, or 2.0 g/hr).  The plasma vitamin C levels increased rapidly, remained elevated throughout the 8 hour infusion period, and decreased rapidly upon termination of the infusion.

Figure 2.  Plasma vitamin C levels of four subjects infused at a dosage of 2 g/hr.  The plasma vitamin C levels increased to 162 to 290 μg/ml during the infusion period, but decreased rapidly upon termination of the infusion.  Vitamin C levels for each subject are shown as individual data points and the mean of these four data points at each time is shown as a line.

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Visualization of Formylpeptide Receptor Recovery

April 17, 2012

This study used our autoradiographic method of visualizing locations and amounts of formylpeptide chemotaxis receptors on the surface of rabbit peritoneal neutrophils (heterophils).  We first exposed the PMN to unlabeled formylpeptide to cause down-regulation of the receptor-ligand complexes and then allowed receptor recovery to occur over a 120 min time course.  We used iodinated formylhexapeptide to tag the re-expressed receptors and visualized this using autoradiography.  This was the first time that chemotaxis receptor recycling had been observed and the sites of receptor reinsertion had been localized.

We initially tried to do these studies using fluorescent labeled peptides, but the labeling interfered with the biological activity of the peptide and  was also so weak that it was not possible to obtain useful information. Studies like this may now be more feasible using fluorescent labeled chemoattractant, but the fluorochromes are still quite large compared to the formylated tri- or hexapeptide chemoattractants.  As a result, steric interference with the receptor or surrounding membrane components may occur.  The problem we had with weak signal can probably be overcome using image intensifying video or other digital techniques.

Below is the full paper and a link to the PDF:

Visualization  of Formylpeptide Receptor Recovery on

Rabbit Peritoneal Neutrophils

Robert J. Walter and Wayne A. Marasco

Department of Anatomy

University of Illinois at Chicago

P.O.Box 6998

Chicago, IL  60680

Department of Pathology

University of Michigan Medical School

Ann Arbor, MI  48109

Running Title: Formyl Peptide Receptor Recovery

Keywords - chemotaxis,  leukocytes, formylpeptide, receptors, recycling, down-regulation , up-regulation, reinsertion, plasma membrane, cell surface

ABSTRACT

We have used light microscope autoradiography to visualize binding of the formylhexapeptide, N-formyl-norleucyl-leucyl-phenylalanyl-norleucy-¹²⁵I-tyrosyl-lysine to rounded and polarized rabbit polymorphonuclear leukocytes that had been pre-treated with unlabeled formylpeptide. These cells possess receptors known to bind with high specificity and great avidity to the chemotactic formylpeptides. Cells adherent to glass slides were exposed to 10 nM unlabeled hexapeptide at 37ºC for 30 min.  Slides were then rinsed in 4ºC buffer to remove unbound peptide and then placed in buffer at either 4ºC or 37ºC for up to 120 min to allow recovery of hexapeptide receptors on the cell surface.  Upon completion of the proscribed recovery period, slides and adherent PMN were rinsed in 3 changes of buffer, exposed to I¹²⁵-labeled hexapeptide (5 nM) at 4ºC for 15 min, rinsed in buffer, and then fixed.  Labeled peptide was then detected autoradiographically to quantify and localize formylpeptide receptors on the cell surface.  Initial reinsertion (10 min or less recovery) was uniform across the cell surface, but later reinsertion or redistribution of inserted receptors occurred such that a non-uniform distribution of receptors was seen.  Increased receptor numbers (2.6 to 3.4 times higher) were seen on the anterior half of cell than the posterior half.  Non-pretreated or unchallenged cells showed no such asymmetric disposition of receptors.  The total number of receptors expressed at each recovery time was approximately equal for rounded and polarized PMN indicating that polarized cells were not a special subpopulation of cells expressing an unusually small or large number of receptors.  

 

INTRODUCTION

   

Polymorphonuclear leukocytes (PMN) possess cell surface receptors that specifically bind certain soluble bacterial factors and their analogs, the synthetic  formylmethionylpeptides.  PMN must continually reassess the surrounding milieu in order to detect formylpeptide concentration gradients, to initiate cell migration, and to perpetuate directional locomotion.  Since these activities are mediated by the binding of chemoattractant to specific cell surface receptors, free receptors must either be continually added to the cell membrane during locomotion or receptors must be freed from bound ligand in some fashion.  This could be accomplished by any of several mechanisms including de novo receptor synthesis, insertion of receptors from intracellular pools, or cleavage of ligand from its receptor.  In the latter case, cleavage could occur either on the cell surface or intracellularly with free receptors subsequently returned to the cell surface.  As a result of such processes, free receptors might appear either randomly on the cell membrane or in restricted locations of the cell surface.  The sites of reappearance of free receptor on the cell surface during chemotaxis may play a significant role in modulating the cell`s ongoing response to chemotactic stimuli. Such modulation may either aid or hinder further cellular adaptation to changing concentrations of chemoattractant.  We have examined recovery of cell surface receptors for the formylpeptide chemoattractant, N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine on rabbit peritoneal leukocytes initially forced to down-regulate their free hexapeptide receptors.  We have studied this recovery morphologically and determined the locations of reappearance of free receptors on the cell surface.

METHODS AND MATERIALS

Isolation of Rabbit Peritoneal Cells

New Zealand white rabbits were injected intraperitoneally with 100 ml of 0.1% oyster glycogen in sterile saline.  After 16 h, the rabbits were injected with sterile saline (50 ml) and the peritoneal exudate drawn off. Exudate was collected in a siliconized flask, chilled on ice, centrifuged, and the cells resuspended in buffer containing 140 mM NaCl, 10 mM KCl, 10 mM HEPES, 5 mM glucose, 1 mM MgSO₄, 0.2 mM CaCl₂, and 2 mg/ml bovine serum albumin, pH 7.4 (HBS). Cells were then stored at 4ºC until use.  Cell preparations contained more than 95% polymorphonuclear leukocytes with the remaining cells being predominantly monocytes.

Exposure to Iodinated Hexapeptide

PMN suspended in 50 µl buffer were allowed to settle and adhere to acid-cleaned glass microscope slides in a humidified chamber.  After 5 min at 37ºC, 50 µl of 20 nM unlabeled hexapeptide (10 nM final concentration) was added to each slide and the slides with adherent cells incubated further at 37ºC for 30 min.  Slides were then rinsed in 4ºC buffer and then placed in buffer at either 4ºC or 37ºC for 0, 5, 10, 20, 40, 60, or 120 min to allow recovery of hexapeptide receptors on the cell surface.  Upon completion of the proscribed recovery period, slides and adherent PMN were rinsed in 3 changes of buffer, exposed to I¹²⁵-labeled hexapeptide (5 nM) at 4ºC for 15 min, rinsed in buffer, and then fixed in a solution containing 1.5% glutaraldehyde, 1.0% paraformaldehyde and 0.1 M cacodylate.

Autoradiography and Quantitative Methods

Cells were fixed overnight, rinsed in 0.1 M cacodylate buffer, in Dulbecco's modified Eagle's medium, in cacodylate buffer again, and then dehydrated in graded ethanols to 80% ethanol.  Cells were then rehydrated, and slides dipped in Kodak NTB-2 emulsion (diluted 1:5 with distilled water), air dried, and stored for 4 days at 4ºC in the dark. Exposed autoradiographs were developed using Kodak D-19, fixed, stained in eosin and cresyl violet, and coverglasses affixed using Permount.

Cells were examined and photographed using a Nikon Optiphot microscope with an Olympus camera.  Phase-contrast optics were used to determine cell morphology and dark-field optics to visualize and count silver grains associated with the cells. Grains associated with 50 cells were counted for each experimental group and duplicate samples were run for each group. For anteroposterior grain distributions, polarized PMN were transected by a line drawn midway between the leading edge and trailing uropod tip.  Grains over each half of the cell were then counted.  Groups were compared using Mann-Whitney U tests (Instat, GraphPad software).

RESULTS

Time Course of Receptor Recovery

PMN not pre-treated with cold hexapeptide exhibited a large capacity for binding I¹²⁵-labeled hexapeptide (Figure 1).  This capacity did not change significantly during the time course of the experiment. Cells pre-treated with 10 nM cold hexapeptide and then allowed to further incubate in buffer at 4ºC, did not display appreciable amounts of hexapeptide binding during the 120 min recovery time course.  However, cells pre-treated with cold peptide and then incubated at 37ºC in buffer gradually recovered most of their surface receptors for formyl hexapeptide by the end of the 120 min time course (72% recovery).  Reappearance of hexapeptide binding capacity by rabbit peritoneal PMN occurred rapidly for 10 min but then slowed to a new rate for the remainder of the experiment.  Binding of I¹²⁵-hexapeptide to PMN was negligible (1-5%) in all control groups exposed simultaneously to 5 nM I¹²⁵-hexapeptide and 5 µM cold, unlabeled hexapeptide.

Receptor Distribution During Recovery

During the initial 10 min of receptor recovery at 37ºC, hexapeptide receptors were distributed somewhat uniformly on the cell surface of motile or polarized PMN (Figure 2). However, as the time of recovery progressed, increasing numbers of receptors appeared on the front half of each cell and fewer on the rear half (Figure 3).  Cells not pre-treated with chemoattractant yet incubated at 37ºC for 60 min displayed a nearly uniform distribution of hexapeptide (Figure 4a, b).  On the other hand, hexapeptide pretreated cells recovered at 4ºC for 60 min (Figure 4c, d) and controls for nonspecific binding (Figure 3e, f) showed very few cell associated grains.

Grain counts were also performed on polarized PMN as seen in Table I. Polarized cells were identified using phase contrast optics and their overall length measured using an eyepiece micrometer.  This length was halved and the micrometer line corresponding to this midpoint used to distinguish the anterior or front half of the cell from the posterior or rear half of the cell.  This midpoint was usually found to lie at the posterior boundary of the nucleus. Grains over each half were counted and the ratio of grains on the front:rear of the cells calculated.  Cells not pretreated with hexapeptide and cells pretreated with hexapeptide and recovered for 10 min at 37ºC exhibited a nearly equal anteroposterior distribution of hexapeptide receptors. Pretreated cells recovered for longer periods of time (i.e., 40 and 60 min at 37ºC) exhibited a significantly increased (p<0.001) number of receptors on the front half of each cell compared to the number seen on the posterior half.  Few grains were counted on pretreated cells recovered at 4ºC.  Also note that the total numbers of grains seen on motile cells (front + rear) are very similar to the numbers of grains seen on rounded cells at each stage of recovery (Figure 1).

DISCUSSION

Recovery visualized; goes nearly to completion; occurs only at 37ºC; nonspecific binding negligible; very little recovery at 4ºC;  2 rates of recovery seen --- rapid initially, slower later;  2 possible mechanisms of reinsertion or 2 different sources of receptors available for membrane insertion.

   Initial reinsertion appears uniform; later reinsertion or redistribution of inserted receptors appears non-uniform with tendency toward anterior half of cell; non-pretreated or challenged cells show no predisposition of receptors.

   Total number of receptors expressed at each recovery time approx. equal for rounded and polarized PMN; polarized cells are not a special subpopulation of cells expressing an unusually

small or large number of receptors.

   After recovery is complete, the distribution of receptors may become uniform as seen in the not pretreated group.

ACKNOWLEDGMENTS

   This work was supported in part by grant #85-26 from the American Cancer Society, Illinois Division, Inc. (RJW) and by NIH grants # (WAM).

TABLE I

Grain Counts on Polarized PMN during Formylpeptide Receptor Recovery

Treatment                                   Grains (mean ± SEM)               Ratio                 p**

                                                PMN Front*    PMN Rear*  (Front/Rear)

___________________________________________________________________

Not Pretreated - 37ºC             15.8 ± 1.0        13.6 ± 1.8          1.16               ---

Pretreated/ Recovered

      

 0 min at 37ºC                          0.40 ± 0.2        0.30 ± 0.1          ----                ---

  

10 min at 37ºC                         2.35 ± 0.5        2.35 ± 0.5          1.00               >0.05

40 min at 37ºC                         7.50 ± 1.4        2.85 ± 0.5          2.63               <0.001**

60 min at 37ºC                         10.3 ± 1.7        3.00 ± 0.4          3.43               <0.001**

   

10 min at 4ºC                           1.3 ± 0.3          0.7 ± 0.2            ----                   ---

60 min at 4ºC                           0.7 ± 0.3          0.0 ± 0.0            ----                   ---

* Polarized PMN were transected by a line midway between the leading edge and trailing uropod tip.  Grains over each half of the cell were then counted.

Duplicate data points from 2 experiments are summarized.  Fifty cells were counted for each data point in each experiment.

** Front vs Rear ratios compared to “Not Pretreated – 37ºC” group using Mann-Whitney U tests.

REFERENCES

ANDERSON, R. and NIEDEL, J. 1984.  Processing of the formylpeptide receptor by HL-60 cells.  J. Biol. Chem. 259: 13309-13315.

BERLIN, R.D. and OLIVER, J.M. 1982.  The movement of bound ligands over cell surafces. J. Theor. Biol. 99:69-80.

CRESSIE, N.A.C., SHEFFIELD, L.J., and WHITFORD, H.J. 1984.  Use of the one sample t-test in the real world. J. Chron. Dis. 37: 107-114.

DAUGHADAY, C.C., MEHTA, J., SPILBERG, I., and ATKINSON, J.P. 1985.  Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine:

Chemotaxis, superoxide generation, and binding. J. Immunol. 134: 1823-1826.

DAUKAS, G., LAUFFENBURGER, D.A., and ZIGMOND, S. 1983.  Reversible pinocytosis in polymorphonuclear leukocytes. J. Cell Biol. 96:1642-1650.

FERTUCK, H.C. and SALPETER, M.M. 1974.  Sensitivity in electron microscope autoradiography for I-125. J. Histochem. Cytochem. 22: 80-87.

GALLIN, J.I. 1984.  Human neutrophil heterogeneity exists, but is it meaningful? Blood 63: 977-983.

GALLIN, J.I., SELIGMANN, B.E., and FLETCHER, M.P. 1983.  Dynamics of human neutrophil receptors for the chemoattractant f-met-leu-phe. Agents Actions (Suppl.) 12: 290-308.

GALLIN, J.I. and SELIGMANN, B.E. 1984.  Neutrophil chemoattractant fMet-Leu-Phe receptor expression and ionic events following activation. Contemp. Top. Immunobiol. 14: 83-108.

GOLDMAN, D.W. and GOETZL, E.J. 1984.  Heterogeneity of human polymorphonuclear  leukocyte receptors for leukotriene B4. Identification of a subset of high affinity receptors that transduce the chemotactic response. J. Exp. Med. 159: 1027-1041.

HARVATH, L. and LEONARD, E.J. 1982.  Two neutrophil populations in human blood with different chemotactic activities: Separation and chemoattractant binding. Infec. Immun. 36: 443-449.

JESAITIS, A.J., NAEMURA, J.R., PAINTER, R.G., SCHMITT, M., SKLAR, L.A., and COCHRANE, C.G. 1982.  The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: Subcellular fractionation studies. J. Cell. Biochem. 20: 177-191.

JESAITIS, A.J., NAEMURA, J.R., PAINTER, R.G., SKLAR, L.A., and COCHRANE, C.G.  1983.  The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies. J. Biol. Chem. 258: 1968-1977.

JESAITIS, A.J., NAEMURA, J.R., SKLAR, L.A., COCHRANE, C.G., and PAINTER, R.G.  1984.  Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: Formation of high-affinity ligand-receptor complexes in transient association with cytoskeleton. J. Cell Biol. 98: 1378-1387.

KOO, C., LEFKOWITZ, R.J., and SNYDERMAN, R. 1982.  The oligopeptide chemotactic factor  receptor on human polymorphonuclear leukocyte membranes exists in two affinity states. Biochem. Biophys. Res. Comm. 106: 442-449.

MACKIN, W.M., HUANG, C.-K., and BECKER, E.L. 1982.  The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities. J. Immunol. 129: 1608-1611.

MARASCO, W.A., PHAN, S.H., KRUTZSCH, H., SHOWELL, H.J., FELTNER, D.E., NAIRN,  R., BECKER, E.L., and WARD, P.A. 1984.  Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J. Biol. Chem. 259: 5430-5439.

NIEDEL, J.E. and CUATRECASAS, P. 1980.  Formyl peptide chemotactic receptors of leukocytes and macrophages. Curr. Top. Cell. Regul. 17: 137-170.

PEREZ,H.D., ONG, R.R., and ELFMAN, F. 1985.  Removal or oxidation of surface membrane sialic acid inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis. J. Immunol. 134: 1902-1908.

RAMSEY, W.S. 1974.  Retraction fibers and leucocyte chemotaxis. Exp. Cell Res. 86:184-187.

SALPETER, M.M., FERTUCK, H.C., and SALPETER, E.E. 1977.  Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters. J. Cell Biol. 72: 161-173.

SCHIFFMAN, E. and GALLIN, J.I. 1979.  Biochemistry of phagocyte chemotaxis. Curr. Top. Cell. Regul. 15: 203-261.

SELIGMANN, B., T.H. CHUSED, and J.I. GALLIN. 1984.  Differential binding of chemoattractant peptide to subpopulations of human neutrophils. J. Immunol. 133: 2641-2646.

SELIGMANN, B.E., FLETCHER, M.P., and GALLIN, J.I. 1982. Adaptation of human neutrophil responsiveness to the chemoattractant N-formylmethionylleucylphenylalanine. Heterogeneity and/or negative cooperative interaction of receptors. J. Biol. Chem. 257: 6280-6286.

SELIGMANN, B., MELNICK, D.A., MALECH, H.L., and GALLIN, J.I. 1983.  Identification of two subpopulations of neutrophils using the antineutrophil antibody 31D8 and correlation with functional responsiveness. J. Cell Biol. 97: 419a (Abstr.).

SKLAR, L.A., FINNEY, D.A., OADES, Z.G., JESAITIS, A.J., PAINTER, R.G., and COCHRANE, C.G. 1984.  The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil. J. Biol. Chem. 259: 5661-5669.

SNYDERMAN, R. and PIKE, M.C. 1984.  Chemoattractant receptors on phagocytic cells. Ann. Rev. Immunol. 2: 257-281.

SNYDERMAN, R. and PIKE, M.C. 1984.  Transductional mechanisms of chemoattractant  receptors on leukocytes. Contemp. Top. Immunobiol. 14: 1-28.

SOLOMKIN, J.S., COTTA, L.A., BRODT, J.K., and OGLE, C.K. 1984.  Neutrophil dysfunction in sepsis. III. Degranulation as a mechanism for nonspecific deactivation. J. Surg. Res. 36: 407-412.

SOUTHWICK, F.S. and STOSSEL, T.P. 1983.  Contractile proteins in leukocyte function. Semin. Hemat. 20: 305-321.

STOSSEL, T.P., HARTWIG, J.H., YIN, H.L., SOUTHWICK, F.S., and ZANER, K.S.  The motor of leukocytes. Fed. Proc. 43: 2760-2763.

SULLIVAN, S.J., DAUKAS, G., and ZIGMOND, S.H. 1984.  Asymmetric distribution of the chemotactic peptide receptor on polymorphonuclear leukocytes. J. Cell Biol. 99: 1461-1467.

SULLIVAN, S.J. and ZIGMOND, S.H. 1980.  Chemotactic peptide receptor modulation in polymorphonuclear leukocytes. J. Cell Biol. 85: 703-711.

WALTER, R.J., BERLIN, R.D., and OLIVER, J.M. 1980.  Asymmetric Fc receptor distribution on human PMN oriented in a chemotactic gradient. Nature 286: 724-725.

WALTER, R.J. and MARASCO, W.A. 1984.  Localization of chemotactic peptide receptors on rabbit neutrophils. Exp. Cell Res. 154: 613-618.

ZIGMOND, S.H., SULLIVAN, S.J., and LAUFFENBURGER, D.A. 1982.  Kinetic analysis of chemotactic peptide receptor modulation.  J. Cell Biol. 92: 34-43.

FIGURES

 Figure 1.  Recovery of binding sites for I¹²⁵-hexapeptide on the surface of rabbit PMN.  Cells incubated in buffer at 37ºC for 0 to 120 min (solid circles) subsequently displayed the greatest binding capacity for I¹²⁵-labeled hexapeptide.  Cells pretreated with 10 nM unlabeled hexapeptide for 30 min at 37ºC and then further incubated at 4ºC in buffer (open circles) exhibited very little ability to bind I¹²⁵-labeled hexapeptide.  However, cells pretreated with unlabeled hexapeptide and then further incubated at 37ºC in buffer alone (solid squares) exhibited a gradual increase in receptor-mediated I¹²⁵-hexapeptide binding with time.  There also appeared to be an inflection point at about 10 min of incubation at which the rate of binding recovery decreased.  Mean ± SD, N=50 cells per data point.

Figure 2.  Occurrence and distribution of silver grains associated with PMN after 10 min of receptor recovery at 37ºC.  Phase contrast (left) and corresponding dark-field (right) images of polarized (a, b) and rounded (c, d) cells.  The distribution of grains on polarized cells was generally uniform.  The number of grains on the polarized cell (a, b) seen here is somewhat greater than average for this time point in the recovery sequence, however.  The number of grains seen on the rounded cells seen in 2a and b is more typical of cells recovered for 10 min at 37ºC.  Magnification bar = 10 µm.

Figure 3.  Occurrence and distribution of silver grains associated with PMN after 60 min of receptor recovery.  Phase contrast (left) and corresponding dark-field (right) images of PMN.  After 60 min of recovery at 37ºC, grains were seen predominantly over the anterior half of the polarized cells (a, b) and in abundance but uniformly distributed over rounded cells (c, d).  Hexapeptide pretreated PMN subsequently incubated at 4ºC, however, displayed very few cell-associated grains (e, f).  Magnification bar = 10 µm.

 Figure 4.  Controls for I¹²⁵-hexapeptide binding on rabbit PMN.  Phase contrast (left) and dark-field (right) images of cells not pretreated with unlabeled hexapeptide (a, b) and cells exposed to I¹²⁵-hexapeptide in the presence of 1000-fold excess unlabeled hexapeptide (c, d).  The former treatment represents a positive control demonstrating the large amounts of binding possible with cells that have not been pretreated with unlabeled hexapeptide.  Both polarized and rounded cells labeled heavily under these conditions.  The latter treatment indicated the specificity of the iodinated hexapeptide probe.  There were very few grains evident in such preparations and virtually no grains were cell associated.  Magnification bar = 10 µm.

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Release of Chemotactic Activity by Platelets from Diabetic Rhesus Monkey

April 15, 2012

This is a study done many years ago using a unique colony of rhesus monkeys that had been made diabetic using streptozotocin, a drug that is highly toxic to pancreatic islet cells.  This colony had been maintained on daily insulin injections for many years thereafter and had been studied as a model of human diabetes to great advantage by the Jonasson group.  For the present study, we examined the generation of chemotaxis factors derived from normal and diabetic monkey platelets.  The significance of the study related to the long term vascular and related tissue lesions often seen in human diabetes.

Interestingly, this colony had to be discontinued eventually when the considerable funding required for its ongoing maintenance was lost.  It seemed like a very valuable resource at the time primarily because animal models for long-term effects of diabetes were rare and a primate model was exceedingly rare.  I don't know if such a model even exists at present in view of the great expense of maintaining it and the political incorrectness of using primates for research.  However, diabetes marches on despite political correctness and is now a far greater public health problem in the US than it was when this study was performed.

Below is the full paper and a link to the PDF.

Release of Chemotactic Activity by Platelets from Diabetic Monkeys

Agonist-Induced Release of Chemotactic Activity by  

Platelets Isolated from Diabetic Rhesus Monkey

 

Glenn S. Takimoto,¹Robert J. Walter, James D. Jeffery,

Anne F. Bauman and Olga Jonasson

 

Department of Surgery

University of Illinois College of Medicine

and 

¹ Department of Surgery, Cook County Hospital

Chicago, IL  60612

Send all correspondence to:

Robert J. Walter, Ph.D.

Department of Surgery

Hektoen Institute for Medical Research

627 South Wood St.

Chicago, IL  60612

Running Title: Chemotaxis and Diabetic Platelet Activation

ABSTRACT

Agonist-induced liberation of chemotactic and chemokinetic activity from normal rhesus monkey platelets was characterized and compared to activity liberated from platelets obtained from streptozotocin-treated, insulin-deficient diabetic (STZ-ID) monkeys. The STZ-ID monkeys were severely hyperglycemic with minimal residual c-peptide secretion, hyperglucagonemic, and had elevated glycosylated hemoglobin levels. Fifteen control and 7 STZ-ID monkeys were studied. Platelet-free supernatants isolated from washed, intact platelet preparations incubated with agonist were applied to a microchemotaxis system employing mouse 3T3 fibroblasts as target cells. Using platelets from control monkeys, dose-dependent increases in aggregation response and liberation of chemotactic activity were obtained when thrombin and arachidonic acid were employed as agonists. These responses were attenuated by known platelet activation inhibitors that increase cAMP levels or mimic its cellular actions. When control and STZ-ID monkey groups were compared, thrombin-induced liberation of chemotactic activity was greater with platelets isolated from the STZ-ID group. Arachidonic acid-induced liberation of chemotactic activity was similar for both groups. There were also no differences between control and STZ-ID groups in platelet aggregation responses to either thrombin or arachidonic acid. Supernatants from platelet suspensions exposed to either thrombin or arachidonic acid showed markedly stimulated 3T3 cell chemokinesis, but no differences in agonist-induced liberation of chemokinetic activity were observed when comparing results from control and STZ-ID groups. In summary, we have demonstrated using agonist stimulation of intact, washed platelets that the liberation of chemotactic activity but not aggregation is enhanced with chronic hyperglycemia.

Key Words: chemotaxis, chemokinesis, platelet, rhesus monkey, diabetes, platelet aggregation, 3T3 fibroblasts

               

INTRODUCTION

Platelets from humans with Type I diabetes exhibit increased sensitivity to proaggregatory agents [1-4].  This sensitivity is thought to contribute to the development of macro- and microvascular lesions seen in these patients [5,6].  It was initially postulated that the role of platelets in lesion development was mediated principally through enhanced platelet aggregate formation. However, subsequent studies have suggested that an alteration in the formation and release of platelet-derived bioactive agents may also be involved.  Many platelet products are liberated during agonist-induced activation but their precise functions are only partially understood.  Prostaglandin endoperoxides or thromboxanes and dense granule constituents are known to promote platelet aggregation [7], but little is known about the function of other granule-stored constituents or about lipoxygenase enzyme products of arachidonic acid.  Recent studies by several investigators demonstrate that the alpha granule constituents (platelet-derived growth factor (PDGF), β-thromboglobulin, platelet factor 4) and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid promote migration of a variety of cell types including vascular smooth muscle, endothelial cells [8,9], fibroblasts [10], and neutrophils [11]. Since these substances may be derived from activated platelets they are relevant to vascular lesion development and might also affect host immune response to infections and wound healing.  However, the actual involvement of such platelet-derived substances in the pathophysiology of diabetes remains to be clarified.  

A large colony of streptozotocin-treated insulin dependent (STZ-ID) diabetic rhesus monkeys is available for our study [12].  The progression of disease in this diabetic monkey colony has been systematically followed for several years.  During this time, glycemic and hormonal changes as well as functional and morphological alterations of kidney and retina have been observed.  The character and extent of these alterations closely resemble those seen in the early, non-proliferative stages of human Type I diabetes [12-14].

Our principal aim here was to determine whether platelets from normal and diabetic monkeys differed in their ability to liberate products affecting cell migration.  Washed platelet suspensions were incubated with a variety of agonists, the platelets were removed, and platelet-free supernatants were applied to mouse 3T3 fibroblasts in a microchemotaxis system.  All platelet supernatants stimulated fibroblast chemokinesis (random cellular movement) and moreover, chemotaxis (directed movement).  Furthermore, certain agonists induced the appearance of significantly greater chemotactic activity in platelet suspension supernatants (PSS) from STZ-ID than control monkeys.

Abbreviations used: PSS, platelet suspension supernatants; STZ-ID, streptozotocin-treated insulin dependent; HEPES, N-2-hydroxyethylpiperazine-N-2`-ethanesulfonic acid; DMEM, Dulbecco`s modified Eagle`s medium; EDTA, ethylenediamine tetraacetic acid; platelet-derived growth factor,  PDGF.

METHODS AND MATERIALS

Reagents and biochemicals were obtained as follows: ADP (sodium salt), arachidonic acid, thrombin (human plasma), ± verapamil (sodium salt), indomethacin, acetylsalicylic acid (aspirin), prostaglandin E1, and HEPES (Sigma Chemical Co., St. Louis, MO); 3-isobutyl-1-methylxanthine (Aldrich Chemical Co., Milwaukee, WI); streptozotocin was a generous gift from The Upjohn Co. (Kalamazoo, MI); human c-peptide standard, ¹²⁵I-c-peptide and M1230 guinea pig anti-human c-peptide antiserum (Novo Research Institute, Bagsvaerd, Denmark); beef-pork glucagon standard was a gift from Eli Lilly and Co. (Indianapolis, IN); 30K rabbit anti-beef-pork glucagon antiserum was obtained from Dr. R. Unger, University of Texas Medical School (Dallas, Texas); ¹²⁵I-glucagon was obtained from Cambridge Medical Diagnostics Inc. (Billerica, MA); hemoglobin A1 electrophoresis kit (American Scientific Products, McGaw Park, IL); trypsin, EDTA, Dulbecco`s modified Eagle`s medium (DMEM) (Gibco Supply, Grand Island, NY).

Animals

Age-matched control and STZ-ID rhesus monkeys were studied. Monkeys were made diabetic by intravenous injection of 45-55 mg/kg streptozotocin.  STZ-ID monkeys were severely hyperglycemic and required exogenous insulin (2-15 U/day) to maintain body weight and prevent ketoacidosis. All monkeys were fed standard Purina monkey chow with the STZ-ID group receiving a fruit supplement. (Refer to Table 1 for additional information).

Plasma glucose, immunoreactive c-peptide and glucagon, and glycosylated hemoglobin

Fasting plasma glucose, immunoreactive c-peptide and immunoreactive glucagon, as well as hemoglobin A1 measurements, were obtained from control and STZ-ID monkeys. The STZ-ID monkey group was severely hyperglycemic (337 ± 22 mg/dL) as compared to the control group (66 ± 2 mg/dL). Sufficient exogenous insulin was provided to the STZ-ID monkeys to prevent ketoacidosis and to allow growth in juvenile monkeys and maintain weight in adult monkeys. Indicative of extensive STZ-induced beta cell destruction with minimal residual insulin secretion were measurements of depressed plasma immunoreactive c-peptide levels in the STZ-ID (74 ± 30) as compared to the control (235 ± 42) group under severe fasting hyperglycemia. Plasma immunoreactive glucagon in the STZ-ID group was also elevated, presumably as a result of reduced beta cell secretion of insulin. Moreover, consistently elevated hemoglobin A1 levels reflect the stability of the hyperglycemia in the diabetic monkey group. Similar findings have been reported previously for this monkey colony [12].

Blood collection and isolation of washed platelets

Blood samples were taken from monkeys that were lightly anesthetized with ketamine after an overnight fast. Blood from a femoral vein was drawn into a plastic syringe through a 19 gauge needle, immediately transferred in 3 ml aliquots to siliconized tubes containing 0.5 ml of 0.129 M buffered citrate and 1.25 ml of saline. A washed platelet fraction was prepared by a modification of the method described by Baenziger and Majerus [15]. Briefly, citrated whole blood was centrifuged at 200xg for 10 min at 20ºC to obtain platelet-rich plasma. Prostaglandin E1 was added to the platelet-rich plasma isolate at a final concentration of 1 µg/ml to prevent platelet activation during further preparative procedures. The isolated platelet-rich plasma was centrifuged again at 200xg to remove contaminating erythrocytes, and the resulting supernatant centrifuged at 3000xg for 15 min at 20ºC. The platelet pellet was resuspended in phosphate buffer, pH 6.5, containing 1 µg/ml prostaglandin E1 and centrifuged again. The final pellet was suspended in serum free Dulbecco`s modified Eagle`s medium (DMEM) containing 25 mM HEPES buffer at a final count of 500x10⁶ platelets/ml.

Platelet aggregation and isolation of activation products

Platelet aggregation was measured by a conventional transmission spectrophotometric method. Aliquots (500 µl) of washed platelet suspensions were added to siliconized glass aggregation tubes, warmed to 37ºC for 2 min and placed in an aggregation chamber with stirring for an additional 1 min. Agonist (5 µl) was then added and the resulting aggregation response monitored for 3 min against a blank containing platelet-free, serum free DMEM/HEPES. After 3 min, aggregation and release were terminated by rapidly transferring the experimental sample to a 1.5 ml microfuge tube and pelleting the platelets by microfuge centrifugation for 2 min. Aliquots of the resulting supernatant (PSS) were placed on ice prior to chemotaxis or chemokinesis measurements. In studies examining the effects of platelet activation inhibitors upon agonist-induced aggregation and liberation of chemotactic activity, washed platelet suspensions were preincubated with inhibitor in aggregation tubes for 14 min at 37ºC without stirring. Tubes were then transferred to the aggregation chamber where samples were treated as described above. Control samples not containing inhibitor were also subjected to a 14 min preincubation period.

Cell culture conditions

NIH 3T3 cells were grown in continuous culture in DMEM supplemented with amino acids (2X), glutamine (2X), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. Cells were maintained at pH 7.2 in a water-jacketed C0₂ incubator at 37ºC and harvested from flasks using 0.1% trypsin with 0.2 mM EDTA. They were centrifuged at 500xg for 5 min, resuspended in fresh suspension medium, counted using a hemocytometer, and kept at 4ºC in suspension until used for chemotaxis studies.

Chemotaxis and chemokinesis measurements

Cells harvested by trypsinization were suspended in serum free DMEM/HEPES (10⁶ cells/ml). Supernatants derived from PSS that were undiluted or diluted 1:3 with this medium were placed into the lower wells of a 48 well chemotaxis chamber (Neuroprobe, Cabin John, MD). A polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA) with 5 µm pores was pretreated with 0.001% poly-L-lysine for 10 min, rinsed, dried, and placed over these lower wells. Suspended cells (25 µl; 25,000 cells) were placed into the upper wells and either additional media, platelet buffer, or PSS was also added (25 µl). Chambers were then incubated at 37ºC in a humidified incubator for 4 hours. This incubation was terminated by removing the filter from the chamber, fixing it in methanol, scraping the adherent, non-migrated cells off the top of the filter, and staining the migrated cells on the underside of the filter with Lillie`s hematoxylin. Filters were then dried onto glass slides, mounted, and the cells counted at 400x magnification. Samples were run in triplicate and 5 microscope fields in each well were counted. Heat inactivated fetal calf serum (1%) was used as a positive control (see Table 2).

In experiments designed to measure chemokinesis, PSS were added to upper (25 µl) and lower (29 µl) chambers while suspended 3T3 cells (25 µl; 25,000 cells) were added to the upper chamber. The number of cells penetrating the polycarbonate filter was then measured as described above.

Statistical analysis

Student`s two-tailed t-test was employed for all statistical analyses.                  

.

RESULTS

Animals

Characteristics of the animals employed in the present study are shown in Table 1. Fifteen control (6 male and 9 female) monkeys ranging in age from 7.4 to 21.9 years with a mean (± SEM) age of 11.5 ± 1.2 years, and 7 STZ-ID (2 male and 5 female) monkeys ranging in age from 6.2 to 16.9 years with a mean (± SEM) age of 10.0 ± 1.3 years were studied. The duration of insulin dependence for the STZ-ID group ranged from 1.2 to 10.3 years with a mean ± SEM) duration of 4.4 ± 1.2 years.  

Platelet aggregation and release of chemotactic activity in control monkeys

Thrombin- and arachidonic acid-induced aggregation and release of chemotactic activity from washed platelets isolated from control monkeys was assessed using cultured mouse 3T3 fibroblasts as target cells in a microchemotaxis assay system. Dose-dependent increases in chemotactic activity and aggregation response were obtained for both agonists (Figure 1). Appearance of chemotactic activity was platelet-dependent since no additional activity was obtained when agonist was added directly to PSS incubated with buffer. However, at high thrombin and arachidonic acid concentrations (above 0.5 U/ml and 30 µM, respectively) residual agonist present in the PSS contributed significantly to the chemotactic activity measured. Serial dilution of PSS from either thrombin- or arachidonic acid-stimulated platelets with serum free DMEM/HEPES led to progressive decreases in chemotactic activity. At concentrations of arachidonic acid above 60 µM the aggregation responses became inversely proportional to agonist concentration, giving the aggregation response curve a bell-shaped appearance. 

Chemotactic activity in control PSS incubated with 0.1 U/ml thrombin was inhibited by preincubation with 3-isobutyl-1-methylxanthine, 8-bromo-cAMP and prostaglandin E1, although aspirin had little effect. On the other hand, both 3-isobutyl-1-methylxanthine and aspirin inhibited the liberation of chemotactic substances from arachidonic acid-treated platelets. Inhibition of the thrombin- and arachidonic acid-induced appearance of chemotactic activity in PSS was also accompanied by an inhibition of the corresponding aggregation response.

Comparison of aggregation and release of chemotactic activity in control and STZ-ID monkey groups

Thrombin- and arachidonic acid-induced release of chemotactic activity was then compared using washed platelets from control and STZ-ID monkeys (Table 2). Thrombin-induced appearance of chemotactic activity in PSS was greater with platelets isolated from STZ-ID than control monkeys. Since chemoattractant stimulation of 3T3 cell migration is known to exhibit bell-shaped dose-response curves, we also examined effects of diluting PSS upon measured chemotactic activity. A significant increase in chemotactic activity was obtained with the STZ-ID group upon 1:2 through 1:8 dilutions of PSS. Data obtained for the 1:3 dilution are shown in Table 2.  However, the corresponding aggregation responses recorded for thrombin-stimulated platelets from control and STZ-ID monkeys were not significantly different.  No significant differences were observed in the arachidonic acid-induced appearance of chemotactic activity in undiluted or diluted PSS, or in the corresponding aggregation responses with platelets from control and STZ-ID monkeys (Table 2). Chemotactic activity obtained with PSS from platelets incubated in the presence of buffer alone was similar for platelets isolated from control and STZ-ID monkeys. It constituted 13-24% of the activity derived from platelets incubated in the presence of agonist.

Platelet-derived chemokinetic activity in control and STZ-ID monkeys

In addition to measuring chemotactic (directed) movement of cultured mouse 3T3 cells, we monitored the effects of PSS upon chemokinetic (stimulated but not directed) movement (Table 3). PSS exposed to thrombin (0.1 U/ml) or arachidonic acid (0.67 µM) stimulated 3T3 chemokinesis markedly (2-8X) over baseline levels obtained with buffer alone. No significant differences in chemokinetic activity were obtained with PSS from STZ-ID and control monkey groups, irrespective of which agonist was employed.  Using similar methods to those used in the evaluation of under-agarose chemotaxis assays, values obtained for chemotaxis were reduced by a factor equal to the chemokinesis to yield a “corrected” chemotaxis value (Table 3).

DISCUSSION

In the present study, we have found that: 1) Washed platelet suspensions stimulated with thrombin or arachidonic acid liberated factors that are both chemokinetic and chemotactic for 3T3 fibroblasts. 2) PSS from diabetic monkeys released more chemotactic activity than did control monkey platelets in response to thrombin.  Similarly, previous studies with extracts of whole platelets, platelet-enriched plasma [16], or purified platelet products [9-11] have presented compelling evidence that platelet-derived compounds are capable of inducing chemotaxis or chemokinesis.  Several platelet-derived products liberated consequent to agonist-induced activation are, in fact, known to have chemotactic activity when added in purified form to a chemotaxis assay system.  For example, the alpha granule constituents, platelet factor 4,  PDGF and β-thromboglobulin, are all strongly chemotactic for human and bovine fibroblasts [10].  Similarly, the lipoxygenase metabolite of arachidonic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, which may be produced by platelets in vivo has been shown to be chemotactic for human neutrophils [11] and rat smooth muscle cells [9].  Whether these substances mediate chemotactic activity following platelet activation in vivo has not been determined.

As a step toward understanding the importance of in vivo platelet activation products, we measured the release of chemotactic activity resulting from exposure of isolated, intact platelets to physiologically relevant agonists. Using this approach, agonist-induced release of chemotactic activity could be studied relative to the concomitant activation of other pathways, such as aggregation and granule release.  We first examined the effects of platelet products formed consequent to agonist stimulation of washed platelet suspensions obtained from control monkeys upon fibroblast cell migration.  Dose-dependent increases in chemotactic activity paralleled corresponding increases in aggregation response when either thrombin or arachidonic acid was employed as agonist.  Known inhibitors of platelet activation, such as 3-isobutyl-1-methylxanthine, 8-bromo-cAMP and prostaglandin E1 that increase or mimic cellular cAMP, inhibited both thrombin and arachidonic acid-induced liberation of chemotactic activity and also corresponding aggregation responses.  The cyclooxygenase enzyme inhibitor, aspirin, inhibited arachidonic acid-induced increases in chemotactic activity and aggregation but had no effect on either response to thrombin.  These results suggest that aggregation and also release of chemotactic activity from agonist-activated platelets are both sensitive to antagonists of established platelet activation pathways.

However, when we compared agonist-induced platelet activation in control and STZ-ID monkey groups, it seemed that the pathways mediating aggregation and liberation of chemotactic activity were affected differently in STZ-ID monkeys.  Following exposure of washed platelet suspensions from control and STZ-ID groups to thrombin, greater chemotactic activity was liberated using platelets obtained from the STZ-ID group, whereas aggregation responses were similar for platelets from both groups.  Using identical incubation conditions, we also found that the release of granule-stored ¹⁴C-5-hydroxytryptamine is similar for control and STZ-ID groups [Takimoto et al., unpublished observations].  Since 5-hydroxytryptamine release from platelet granules usually parallels the aggregation response, these data further emphasize that the liberation of chemotactic activity by STZ-ID monkey platelets seems to be independent of aggregation and total granule release.

The enhanced chemotactic activity observed here was attributable primarily to an increase in directed rather than random (chemokinetic) cellular movement (see Table 2).  It was also agonist-specific, since no difference in chemotactic activity or aggregation response was observed between control and STZ-ID groups when arachidonic acid was employed as agonist.  These findings indicate that: 1) a complete assessment of the pathophysiological changes in platelet function cannot be achieved through aggregation and granule release measurements alone, and 2) that the in vitro liberation of platelet-derived proaggregatory and chemotactic activity may be independently regulated.  Further investigation of these platelet properties will require identification of platelet products mediating the chemotactic response and characterization of pathways regulating the liberation of chemotactic activity.

Platelets from humans with Type I diabetes display increased thrombin-induced aggregation and granule release [8,17], although agonist-induced release of chemotactic activity has not been measured.  The reason for the difference in platelet aggregation and granule release characteristics between diabetic humans and our STZ-ID monkey model is unclear.  Hypertension and hyperlipidemia are frequently associated with diabetes in humans [18,19], and have been shown to increase platelet aggregation, granule release and formation of prostaglandin/thromboxane even in non-diabetic subjects [20,21].  However, our STZ-ID monkeys have thus far failed to exhibit hypertension and hyperlipidemia as well as the advanced, proliferative macro- and microvascular changes which frequently arise in long-term diabetic humans [12].

In summary, our findings demonstrate that agonist-induced aggregation and liberation of chemotactic and chemokinetic activity can be measured using intact, washed platelets from rhesus monkey.  We have also shown that thrombin-induced liberation of chemotactic activity is selectively enhanced with chronic hyperglycemia without alteration of the corresponding aggregation response.  We speculate that the enhanced liberation of chemotactic activity from STZ-ID monkey platelets may be related to hyperglycemia, since secondary complications normally associated with diabetes in humans are absent in the diabetic monkeys.  We have recently induced mineralocorticoid/salt hypertension and diet-induced hyperlipidemia in subsets of our control and diabetic monkeys allowing further study of this problem.  The differential effects of STZ-induced diabetes upon aggregation and liberation of chemotactic activity suggest that these platelet actions are independently regulated.  The significance of the increased in vitro release of platelet-derived chemotactic activity to the development of diabetes-related vascular, immune and tissue repair complications requires further investigation.

ACKNOWLEDGMENTS

This work was supported by USPHS grant AM 18284 and an American Diabetes Association, Northern Illinois Affiliate Young Investigator Award. The authors wish to thank John Krewer for excellent technical assistance.

REFERENCES

1. Sagel, J., Colwell, J.A., Crook, L., Laimins, M. (1975) Ann. Intern. Med. 82, 733-738.

2. Halushka, P.V., Lurie, D., Colwell, J.A. (1977) N. Engl. J. Med. 297, 1306-1310.

3. McDonald, J.W.D., Dupre, J., Rodger, N.W., Champion, M.C., Webb, C.D., Ali, M. (1982) Thromb. Res. 28, 705-712.

4. Preston, F.E., Ward, J.D., Marcola, B.H., Porter, N.R., Timperley, W.R. (1978) Lancet 1, 128-130.

5. Robertson, W.B., Strong, J.P. (1968) Lab. Invest. 18, 538-545.

6. West, K.M. (1978) in Epidemiology of Diabetes and Its Vascular Lesions, Elsevier, New York.

7. Zucker, M.B., Nachmias, V.T. (1985) Arteriosclerosis 5, 2-18.

8. Thorgeirsson, G., Robertson, A.L., Cowan, D.H. (1979) Lab. Invest. 41, 51-62.

9. Nakao, J., Ito, J., Kanayasu, T., Murota, S-I. (1985) Diabetes 34, 185-191.

10. Senior, R.M., Griffin, G.L., Huang, J.S., Walz, D.A., Deuel, T.F. (1983) J. Cell Biology 96, 382-385.

11. Turner, S.R., Tainer, J.A., Lynn, W.S. (1975) Nature 257, 680-681.

12. Jonasson, O., Jones, C.W., Bauman, A., John, E., Manaligod, J., Tso, M.O.M. (1985) Ann. Surg. 201, 27-39.

13. Mogensen, C.E., Christensen, C.K., Vittinghus, E. (1983) Diabetes 32, 64-78.

14. Cunha-Vaz, J.G., Abreu, J., Campos, A., Figo, G. (1975) Br. J. Opthamol. 59, 649-656.

15. Baenziger, N.L., Majerus, P.W. (1974) in Methods in Enzymology, (Fleisher, S., Packer, L., Eds.), Biomembranes Part A, Vol. 31, pp.149-155, Academic Press, New York.

16. Gee, A.P., Minta, J.O. (1984) Am. J. Hematol. 17, 29-38.

17. Chmielewski, J., Farbiszewski, R. (1978) Cells 14, 203-205.

18. West, K.M., Erdreich, L.J., Stober, J.A. (1980) Diabetes 29, 501-508.

19. Mogensen, C.E. (1980) Acta Endocrinol. 94 (Suppl. 238), 103-108.

20. Shattil, S.J., Anaya-Galindo, R., Bennett, J., Colman, R.W., Cooper, R.A. (1975) J. Clin. Invest. 55, 636-643.

21. Vlachakis, N.D., Aledort, L. (1979) Atherosclerosis 32, 451-460.

Table 1

Demographics of monkey subpopulations studied

                       

Animal                  # Studied                Age            Gender        ^a Duration of ID  

            Status                                                (range)                                   (range)

_______________________________________________________________

Control                        15             11.5 ± 1.2        9F, 6M

                                                    (7.4 ± 21.9)

       

 STZ-ID                         7             10.0 ± 1.3        5F, 2M             4.4 ± 1.2

                                                                 (6.2-16.9)                                (1.2-10.3)

_______________________________________________________________

STZ-ID = streptozotocin-treated, insulin-dependent. Values for Age (years) and Duration of ID (insulin dependence; years) represent the mean ± SEM.

^a Duration of insulin-dependence

Table 2

Agonist-induced aggregation and release of chemotactic and chemokinetic activity

using washed platelets isolated from control and diabetic monkeys

                                                                                                                             

Animal              Agonist                    Aggregation         Chemotaxis (# cells/400x field)                       

Status                                                                      ^a undiluted         1:3 dilution        N

____________________________________________________________________

Control             Buffer                                0               7.2 ± 1.6              <1              15

STZ-ID                                                     0                8.0 ± 2.7              <1                6

Control             Thrombin                  26.1 ± 3.5        38.4 ± 4.8          14.4 ± 3.3      15

STZ-ID            (0.1 U/ml)                 31.0 ± 7.2      *63.3 ± 9.1      **38.0 ± 8.5        8

Control             Arach. Acid              10.2 ± 2.8       30.0 ± 4.8           15.8 ± 2.5      11

STZ-ID            (0.67 µM)                 13.0 ± 3.9       47.2 ± 7.7           22.1 ± 5.5        5

^b CX                 FCS                         23.2 ± 1.7            ---                      ---             15

 Control            (1.0%)

_____________________________________________________________________

Buffer or agonist (5 µl) were added to washed platelet suspensions (500 µl), and the

aggregation response was recorded over the ensuing 3 min. The aggregation period was

terminated by microfuge centrifugation and the resulting supernatant applied to the

microchemotaxis chamber. All values represent the mean ± SEM. N = number of different

monkeys from which measurements were obtained.

^a Platelet supernatants were used either undiluted or diluted 1:3 with serum-free DMEM/ HEPES media.

^b In chemotaxis control wells, no platelet supernatants were present. Instead, 1% fetal calf serum was added to the lower wells and 3T3 cells in serum-free DMEM/HEPES added to the upper wells. Such control wells were normally present in the 48-well assay described here. N = number of different experiments in which measurements for 1% fetal calf serum controls were obtained.

*   significantly different from control at p< 0.05

** significantly different from control at p< 0.02

Table 3

Agonist-induced release of chemokinetic activity using

platelets isolated from control and diabetic monkeys

                                                                                                                       

Animal              Agonist               Concentration         Chemokinesis             Corrected                   

Status                                                                     # cells/400x field  (n)     Chemotaxis

___________________________________________________________________

 

Control             Buffer                         ---                     4.2 ± 1.3  (8)                 3.0

STZ-ID                                              ---                    12.6 ± 4.1  (5)                   0

Control             Thrombin               0.1 U/ml                34.0 ± 7.7 (13)               4.4

STZ-ID                                                                       27.3 ± 8.7  (6)            *36.0

Control             Arachidonic           0.67 µM                19.4 ± 2.5  (9)               10.6

STZ-ID            Acid                                                   29.9 ± 5.3  (6)             *17.3

____________________________________________________________________

Incubation conditions and preparation of platelet-free supernatants were as 

described in Table 2. 

All values represent the mean ± SEM. 

(n) = number of different monkeys from which measurements were obtained. 

FIGURES

 

Figure 1.  Platelet aggregation and released chemotactic activity in response to thrombin (A) and arachidonic acid (B). Buffer or agonist (5 µl) was added to washed platelet suspensions (500 µl), and the aggregation response was recorded over the ensuing 3 min. The aggregation period was terminated by microfuge centrifugation and the resulting supernatant applied to the microchemotaxis chamber. All values represent the mean ± SEM of data obtained from 3-15 control monkeys.

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