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.

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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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