Mechanism of Cancer Chemotaxis Defect

May 2, 2012

When we were studying the chemotaxis defect known to exist in leukocytes from cancer patients, we looked at formylpeptide receptor dynamics in considerable detail.  We examined receptor binding, down-regulation, recovery, and receptor-mediated pinocytosis using rabbit peritoneal PMN, human peripheral PMN, and human peripheral monocytes.  The cells were either obtained directly from cancer patients or were from normal subjects.  Cells from normal subjects were pretreated with serum that had been obtained from cancer or normal patients.

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

 Mechanism of Cancer Chemotaxis Defect

 

Mechanism of the Cancer-Related Leukocyte Chemotaxis Defect: 

Formylpeptide Receptor Modulation and Pinocytosis

Amelia H. Janeczek, PhD^1,3, Pierson J. Van Alten, PhD¹, Hernan M. Reyes, MD²,

and Robert J. Walter, PhD²

¹  Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL  60612

²  Department of Surgery, Cook County Hospital, Hektoen Institute for Medical Research, Chicago, IL  60612

³  Present address:  Department of Biochemistry, School of Medicine, Boston University, Boston, MA  02118-2394

Address all correspondence to:

Robert J. Walter, PhD

Department of Surgery, Room 905

Hektoen Institute for Medical Research

625 South Wood Street

Chicago, IL  60612

Telephone:        (312) 633-7237/8717           FAX:       (312) 738-3102

Running Title:  Mechanism of Cancer Chemotaxis Defect

Keywords:  formylpeptide, receptors, cancer, chemotaxis, endocytosis

SUMMARY

Monocyte chemotaxis is severely depressed in patients with advanced tumors but the cellular basis for this chemotactic defect is not known.  Pretreatment of normal human leukocytes or rabbit peritoneal neutrophils (PMN) with serum from cancer (CA) patients inhibits both monocyte and PMN chemotaxis as compared to leukocytes pretreated with serum from healthy (CT) blood donors.  Using purified fresh CA patient leukocytes or CA serum-treated normal leukocytes, formylpeptide receptor binding and modulation were quantified using radiolabeled formylmethionyl-leucyl-phenylalanine (³H-FMLP).  The cell surface binding of ³H-FMLP at 4°C was significantly reduced in CA serum pretreated rabbit peritoneal PMN, but not in CA serum pretreated human peripheral blood PMN or mononuclear leukocytes as compared to CT serum pretreated cells.  However, in purified mononuclear leukocytes isolated directly from tumor patients, formylpeptide binding was significantly reduced as compared to those of normal subjects.  PMN from tumor patients exhibited no significant difference in this regard as compared to PMN from control subjects.  The time course and dose response curves observed during formylpeptide receptor down-regulation were similar for CT and CA serum pretreated cells.  Subsequent to down-regulation, the recovery of cell surface formylpeptide binding at 37°C was similar in its rate and extent in CT and CA serum pre­treated cells.  However, significant reductions in ³H-FMLP uptake at 37°C were seen in tumor patient PMN, in CA serum pretreated rabbit PMN, and in CA serum pretreated human PMN and mononuclear leukocytes as compared to controls.  Such reductions in initial formylpeptide cell surface binding and in formylpeptide endocytosis may contribute directly to the cancer-associated depression of leukocyte chemotaxis.

INTRODUCTION

      Monocyte and macrophage chemotaxis is severely impaired in patients with advanced tumors, but neutrophil (PMN) locomotion is unaffected (1,2).  This defect in monocyte chemotaxis may have life-threatening consequences since host leukocytes may be unable to adequately repress bacterial and fungal infections or tumor growth.  Implantation of tumor cells or injection of tumor sonicates into mice results in reduced accumulation of macrophages in response to inflammatory stimuli, as well as decreased resistance to bacterial infec­tion (3,4).  Similarly, treatment of normal leukocytes with serum from patients with advanced tumors, conditioned media from tumor cell lines, as­cites fluid, plasma, or urine from tumor-bearing mice suppresses monocyte (and often PMN) polarization and chemotaxis (2,5‑9).  This chemo­taxis defect is evident in animals and patients bearing any of a large number of tumors and is seen in response to several quite different classes of che­moattractants (for review see 6,10,11).  Many studies have shown that monocyte chemo­taxis returns to normal after the surgical removal of tumor or after treatment with chemotherapy or radiotherapy (12,13).  Taken together, this suggests that tumors may be producing or causing the host to produce an inhibitor of leukocyte chemotaxis (7,14). 

A soluble inhibitor of monocyte chemotaxis, rendered inactive by treat­ment with antibody directed against the retroviral envelope protein p15E, has been found in tumor patient effusions (15,16) and in serum from patients with head and neck cancer (CA) (17).  This inhibitor or its synthetic ana­logues may cause decreased monocyte polarization in response to chemoattract­ant (8,16), alterations in formylpeptide receptor expression (18,19), suppression of the respiratory burst (20), and inhibition of protein kinase C-related cell functions (21).  It was hypothesized in the present study that the chemotactic defect in tumor patient monocytes or in CA serum-treated leukocytes resulted from alterations in reception or transduction of the signal for chemotaxis.  To examine this possibility, formylpeptide chemoattractant receptor binding was examined in leuko­cytes isolated from tumor patients, in CT or CA serum pretreated human leuko­cytes isolated from normal subjects, and in CT or CA serum pretreated rabbit peritoneal PMN. 

           

Significant reductions in formylpeptide binding were observed in purified tumor patient mononuclear leukocytes as well as in CA serum pretreated rabbit peritoneal PMN.  Chemoattractant uptake at 37°C was significantly reduced in CA serum pretreated rabbit PMN, human PMN, human mononuclear leukocytes, and in tumor patient PMN.  These alterations in surface binding and internalization of formylpeptide may contribute to the depression of chemotaxis exhibited by these cells. 

Abbreviations used: 

BSA, bovine serum albumin; CA, cancer; CT, control; DMSO, dimethylsulfoxide; EDTA, edetic acid; FMLP, N-formyl-methionyl-leucyl-phenylalanine; HBS, HEPES buffered salt solution; HBSS, Hanks' balanced salt solution; HEPES, N-2-hydroxyethylpiperazine-N-2'-ethanesulfonic acid; MLCK, myosin light chain kinase; PMN, polymorphonuclear leukocyte.

                                               METHODS AND MATERIALS

Buffers

For most of the studies described here, HEPES-buffered saline (HBS) containing 140 mM NaCl, 10 mM KCl, 10 mM N-2-hydroxyethylpiperazine N-2-eth­anesulfonic acid (HEPES), 5 mM glucose, and 2 mg/ml bovine serum albumin (BSA), pH 7.4 was used.  For rabbit peritoneal PMN, Hanks' balanced salt solution (HBSS) containing 10 mM HEPES, 5 mM glucose, 136 mM NaCl, 5 mM KCl, 373 nM Na₂HPO₄, 734 nM KH₂PO₄, pH 7.2 was used. 

Rabbit Peritoneal Neutrophils

Glycogen-elicited peritoneal PMN were collected from rabbits in heparin-containing tubes on ice, centrifuged at 500 x g for 10 minutes, and washed in HBSS containing 2 mM EDTA.  Contaminating erythrocytes were removed by brief hypotonic or ammonium chloride lysis, cells were washed in HBSS supplemented with 0.2 mM CaCl₂ and 1 mM MgSO₄, and kept on ice until use.  These cell preparations consisted of >95% PMN.

Isolation and Purification of Human Leukocytes

Peripheral venous blood samples were obtained with informed consent from healthy adult volunteers and from patients admitted to Cook County Hospital for diagnosis and treatment of primary head and neck tumors.  Patients included in this study were not receiving chemo­therapy, radiotherapy, or medication at the time of sample collection.  Blood was collected by venipuncture in sterile EDTA-containing tubes and leukocytes isolated by a modification of the method of Boyum (22).  Erythrocytes were gravity-sedimented at room temperature by the addition of pyrogen-free dextran (200 kD) to a final concentration of 1.25%.  The leukocyte-rich plasma was diluted with an equal volume of HBS with EDTA, layered onto a cushion of Lymphocyte Separation Medium (Organon Teknika Corp, Durham, NC) and centrifuged at 500 x g for 5 min at room temperature.  Mononuclear leukocytes at the interface of the discontinuous gradient were collected, diluted with HBS containing EDTA and 2.5% dextran, and centrifuged at 250 x g for 5 min at room temperature.  The plate­let-rich supernatant was removed and this washing procedure repeated twice.  The PMN pellet was resuspended in HBS with EDTA and centrifuged at 5000 x g in a microcentrifuge for 2 seconds at room temperature.  Contaminating erythrocytes were removed by brief hypotonic lysis and the leukocytes washed three times in HBS with EDTA.  Mononuclear leukocytes and PMN were finally resuspended in HBS containing divalent cations and kept on ice until use.

Serum Pretreatment

Blood samples from healthy adult donors and patients with head and neck tumors were collected by venipuncture and allowed to clot overnight at 4°C.  Serum was collected by centrifugation and stored frozen in aliquots at -80°C until use.  Purified human peripheral blood PMN or mononuclear leukocytes were pretreated by incubation with 10% human serum in HBS with EDTA at 37°C for 30 min in siliconized glass test tubes.  The cells were then centri­fuged at 600 x g for 3 min at room temperature and resuspended in HBS with divalent cations at 4°C. 

Chemotaxis Assays

     N-formyl-methionyl-leucyl-phenylalanine (FMLP; Peninsula Laboratories, Belmont, CA) in concentrations ranging from 0.1 to 100 nM was placed in the bottom wells of a 48 well chemotaxis chamber (Neuroprobe, Cabin John, MD) and covered with a 5 μm pore size polyvinylpyrrolidone-free filter (Nucleopore Corp, Pleasanton, CA) as described previously (10,19).  Briefly, control and CA serum pretreated human PMN or purified mononuclear leukocytes (2-7 x 10⁴ cells) or rabbit peritoneal PMN (2 X 10⁴ cells) were loaded into the upper wells and the chambers were incu­bated at 37°C for 2 hours.  The filters were removed, fixed in methanol, stained, rinsed, dried, and mounted on glass slides using Permount.  Assays were quantitated by counting the number of cells in 5 contiguous 40X microscope fields (23).

³H-FMLP Binding and Uptake Studies

Mononuclear leukocytes (350,000/ 100 μl) or PMN (1 X 10⁶/ 100 μl) were allowed to adhere to acid-cleaned 12 mm diameter round glass coverslips in a humidified chamber at 37°C for 10 minutes.  Control or CA serum was added to each coverslip to a final concentration of 10%, and incubation continued for 30 minutes at 37°C.  Serum was removed by washing the coverslips in HBSS and these preparations studied as described below.  Cell viability remained greater than 90% throughout these experiments.  No cell loss was detected by visual inspection or cell counts of coverslips, and buffer pH was maintained between 7.4 and 7.6.

Baseline FMLP Receptor Binding at 4°C

Adherent, serum pretreated cells on coverslips were rinsed thoroughly in cold HBSS, then incubated in 75 μl of 20 nM ³H-FMLP (58 Ci/mmole; New England Nuclear, Boston, MA) for 60 min at 4°C.  The coverslips were washed briefly in 2 changes of fresh HBSS, immersed in scintillation cocktail (Biofluor; New England Nuclear, Boston, MA), and cell-associated radioactivity determined by scintillation counting (Tm Analytic Inc., Elk Grove Village, IL). 

Formylpeptide Receptor Down-Regulation

To establish a concentration curve for FMLP-induced receptor down- regulation, coverslip-adherent rabbit PMN were pretreated with either CT or CA serum, incubated with varying concentrations of unlabeled FMLP (0.1 - 20 nM) for 20 min at 37°C, rinsed thoroughly in fresh cold buffer, and then exposed to 20 nM ³H-FMLP for 60 min at 4°C.  Baseline cell-associated ³H-FMLP levels were determined in coverslip-adherent cells that had not previ­ously been exposed to unlabeled FMLP.

Receptor down-regulation over a 20 min time course was studied for cells exposed to 5 nM unlabeled FMLP at 37°C.  Subsequently, coverslips were rinsed in cold HBSS, and cell surface formylpeptide receptor expression was assessed using ³H-FMLP as described above.

Formylpeptide Receptor Reexpression on the Cell Surface

Adherent, serum pretreated cells were incubated with 5 nM unlabeled FMLP at 37°C for 20 min to down-regulate formylpeptide receptors, rinsed well with 4 changes of fresh HBSS for 10 min on ice to permit dissociation of surface-bound FMLP, and then further incubated in HBSS for 0 to 60 min at 37°C to allow receptor reexpression on the cell surface.  After these manipulations, the coverslips were rinsed well in cold HBSS, and cell surface formylpeptide receptor expression assessed as described above.

³H-FMLP Uptake

Control and CA serum pretreated adherent cells were incubated with 75 μl of 20 nM ³H-FMLP at 37°C in a humidified chamber for times ranging from 0 to 60 minutes.  At each time point, coverslips were washed in cold HBSS, immersed in BioFluor, and cell-associated radioactivity determined by scintillation counting. 

Statistical Evaluation of Data

Samples were run in triplicate and means of these triplicate groups were compared using Student's paired or unpaired t-tests.  Probability values less than 0.05 were considered significant (24).

RESULTS

Chemotaxis is Reduced in CA Patient Leukocytes and after Pretreatment of Normal Leukocytes with CA Serum

Mononuclear leukocytes from CA patients and PMN or mononuclear leukocytes pretreated with serum showed reduced chemotaxis in response to FMLP as compared to normal leukocytes or to cells similarly pretreated with CT serum.  Since this phenomenon has been described in detail elsewhere (6,7,10,11), statistics descriptive of the samples used here will only be mentioned.  Leukocytes isolated from 6 different CA patients, 17 different CA serum samples, and 8 different CT serum samples were employed.  On the average chemotaxis was reduced in CA mononuclear leukocytes (64%, 6 trials), in CA serum pretreated rabbit peritoneal PMN (40%, 6 trials), in CA serum pretreated human PMN (24%, 24 trials), and in CA serum pretreated human mononuclear leukocytes (60%, 34 trials).  Relative to that seen with CT serum, CA serum alone exhibited no significant chemotactic activity for either PMN or mononuclear leukocytes. 

Initial ³H-Formylpeptide Binding on the Cell Surface at 4°C

Cancer serum pretreated rabbit PMN bound an average of 10% less ³H-FMLP than cells similarly treated with CT serum (p<0.01; paired t-test; Figure 1A).  When human PMN and mononuclear leukocytes from normal controls were studied, no differences in ³H-FMLP binding in CT as compared to CA serum pretreated cells were observed.  In contrast, mononuclear leukocytes isolated from head and neck CA patients bound 42% less ³H-FMLP (p<0.05; Figure 1B), whereas CA patient PMN showed no significant differences in ³H-FMLP binding.

    The specificity of ³H-FMLP binding was tested by exposing coverslip-adherent PMN or mononuclear leukocytes to 20 nM ³H-FMLP in the presence of excess (20 μM) unlabeled FMLP.  Non-specific binding ranged from 1.6% to 5.3% of total ³H-FMLP binding in the experiments reported here and has been subtracted from the total binding to give receptor-specific binding.  Calculations of formylpeptide receptor numbers indicated approximately 43,000 as compared to 36,000 receptors per cell for rabbit PMN pretreated with CT as compared with CA serum.  Similar numbers of formylpeptide receptors on rabbit PMN have been reported elsewhere (25,26).

Formylpeptide Receptor Down-Regulation is not Altered by CA Serum

     1.  Formylpeptide Concentration Curve

The response of CT or CA serum pretreated rabbit PMN to challenge with unlabeled formylpeptide at 37°C was assessed over a range of FMLP concentra­tions (Figure 2).  In the absence of FMLP (i.e., 0 nM in Figure 2), there was no significant difference in ³H-FMLP binding between CT and CA serum pretreat­ed PMN.  Note that the preparation procedure for cells at this data point differed from that described for initial formylpeptide binding (previous section).  After serum treatment, an additional 20 min buffer incubation at 37°C and 10 min buffer incubation at 4°C was employed.  This extended incuba­tion time after serum treatment may have affected the FMLP binding on the cell surface such that a significant difference (as noted in previous section) no longer existed between the CT and CA serum pretreated groups.  The amount of ³H-FMLP binding varied inverse­ly with the concentration of chemoattractant used during preincubation.  With 5 nM unlabeled FMLP, ³H-FMLP counts were 27% and 25% of baseline for CT and CA sera pretreated cells, respectively.  Treatment with higher concentrations of unlabeled chemoattractant did not reduce ³H-FMLP binding beyond the level seen with 5 nM FMLP.  At each concentration of unlabeled FMLP used, ³H-FMLP binding was similar in the CT and CA serum pretreated groups.  Since a substantial reduction of cell-associated ³H-FMLP was obtained with 5 nM unlabeled FMLP, further down-regulation studies were performed using this concentration.

2.      Down-Regulation Time Course

Figure 3 shows that CA serum pretreated rabbit PMN not exposed to unla­beled FMLP (i.e., 0 time) showed 14% less ³H-FMLP binding than the correspond­ing CT serum pretreated group (p<0.02).  With increasing incubation time in unlabeled chemoattractant, cell surface ³H-FMLP binding decreased.  Clearance of receptors was rapid, with more than 50% of the original receptor-mediated binding lost after 2 min of exposure to unlabeled FMLP.  The majority of receptors (78% in CT serum pretreated cells and 75% in CA serum pretreated cells) were cleared within 5 min after initial exposure to unlabeled FMLP.  After 20 min of incubation in unlabeled FMLP, only 9% of original cell surface binding remained in both groups.  No additional significant differences were noted between CT and CA serum pretreated groups during the 20 min down-regula­tion time course. 

Formylpeptide Receptor Reexpression is not Altered by CA Serum

Coverslip-adherent rabbit PMN pretreated with CT or CA serum were first exposed to 5 nM unlabeled FMLP for 20 min, rinsed, and then further incubated in buffer for up to 60 min at 37°C to allow receptor reexpression on the cell surface.  Cells exposed to buffer alone at 37°C instead of 5 nM unlabeled FMLP showed a uniform, high level of ³H-FMLP binding throughout the 60 min observa­tion period (i.e., 100% of receptors present on cell surface).  As seen in Figure 4, PMN exposed to 5 nM unlabeled FMLP for 20 min at 37°C showed greatly decreased cell surface FMLP binding (30% and 32% of initial binding for CT or CA serum pretreated cells, respec­tively).  Upon further incubation in fresh buffer at 37°C, increasing amounts of ³H-FMLP binding were observed.  However, no significant differences in the rate or extent of formylpeptide receptor reexpression were seen in CT as compared to CA serum pretreated PMN.

Uptake of ³H-Formylpeptide at 37°C is Diminished in CA Leukocytes

When leukocytes were incubated with ³H-FMLP at 37°C, the amount of cell-associated peptide increased with time.  For rabbit PMN, a rapid increase in ³H-FMLP accumulation was evident within 2 minutes (Figure 5), but uptake by CT or CA serum pretreated cells did not differ significantly at this time point.  Thereafter, the amount of cell-associated ³H-FMLP gradually increased reaching a peak at 20 minutes, after which time it remained constant.  Cancer serum pretreated cells showed significantly reduced uptake of ³H-FMLP at 10, 20, and 40 min compared to CT serum pre­treated cells (p<0.015).

Serum pretreated human PMN and mononuclear leukocytes exhibited a slower accumu­lation of ³H-FMLP than did rabbit PMN.  During the initial 10 min of exposure to 20 nM ³H-FMLP at 37°C, uptake by CA serum pretreated human leukocytes was not significantly different from that of CT serum pretreated human leukocytes (Figure 6).  Upon further incubation, ³H-FMLP continued to accumulate such that the uptake of ³H-FMLP in the CA serum pretreated leukocytes was 20-45% less than that of the CT serum pretreated leukocytes (10 min, p<0.05; 40 min, p<0.01; 60 min, p<0.04).  Neutrophils isolated from tumor patients and CT subjects (Figure 7) exhibited patterns of ³H-FMLP uptake similar to those observed in serum pretreated leukocytes.  In these samples, uptake of ³H-FMLP was significantly reduced in CA patient PMN at 20, 40 and 60 min (20 min, p<0.001; 40 min, p<0.001; 60 min, p<0.002) as compared to that in CT patient PMN.

DISCUSSION

In the present study, CA patient mononuclear leukocytes as well as rabbit peritoneal PMN, normal human mononuclear leukocytes, and normal human PMN pretreated with 10% CA serum showed consistent reductions in formylpeptide-mediated chemotaxis when compared to leukocytes pretreated with CT serum.  Significant reductions in formylpeptide binding were observed in tumor patient mononuclear leukocytes as well as in CA serum pretreated rabbit peritoneal PMN.  However, formylpeptide receptor modulation, i.e., down-regulation from and receptor reexpression onto the cell surface, in CA serum pretreated leuko­cytes was similar to that seen in CT serum pretreated cells.  Nonetheless, significant reductions in ³H-FMLP uptake at 37°C were seen in CA serum pre­treated rabbit PMN, human PMN, human mononuclear leukocytes as compared to CT serum pre­treated leukocytes and also in tumor patient PMN as compared to PMN from normal subjects. 

Human CA serum inhibits chemotaxis of normal human PMN and monocytes (7) as well as normal guinea pig PMN (27).  Monocytes isolated from CA patient blood display a similar inhibition of chemotaxis, but chemotaxis of PMN from CA patients is not impaired (1,23,28).  The reason for this disparity is not apparent but similar findings were obtained here.  In addition, initial formylpeptide binding was reduced for rabbit PMN pretreated with CA serum as compared to CT serum pretreated cells but no significant differences were noted between CT and CA serum pretreated human leukocytes.  For leukocytes isolated directly from CA patient blood samples, formylpeptide binding was not significantly different for PMN but was significantly reduced (40%) for mononuclear leukocytes as compared to that of leukocytes from normal control subjects.  Similarly, Oostendorp et al. (18) reported decreased formylpep­tide binding by human monocytes and PMN in the presence of the p15E-related peptide, LDLLFL.  If initial formylpeptide binding is reduced, as it is for CA mononuclear leukocytes, then decreased chemotactic responsiveness may be the direct result.  Although CA serum pre­treatment resulted in decreased chemotaxis in human PMN and mononuclear leukocytes, formylpeptide binding was not altered.

To further evaluate the role of formylpeptide receptors in the cancer-associated chemotactic defect, cell surface receptor down-regulation and reexpression were examined.  The results of these studies were consistent with previous reports in which rabbit (29,30) or human (31,32) PMN were used to show that receptor-ligand complexes are removed from the cell surface by internalization result­ing in a net decrease in cell surface formylpeptide receptor expression (32‑35).  There were no significant differences between CT and CA serum pretreated PMN with regard to their formylpeptide receptor complement at any time (except at 0 time) or at any FMLP concentration employed here.  Thus, it seems that the decrease in chemotactic responsiveness in the CA serum pretreated cells is not due to alterations in the receptor down-regulation response or altered clearance of occupied cell surface formylpeptide receptors as seen under these conditions.

     Control and CA serum pretreated rabbit PMN were initially exposed to unlabeled FMLP for 20 min to induce receptor down-regulation, rinsed in fresh buffer, further incubated at 37°C for varying times to permit receptor reex­pression on the cell surface, and the binding capacity of the cells determined using ³H-FMLP.  With increasing incubation time at 37°C, a gradual increase in ³H-FMLP binding occurred but complete receptor reexpression was not observed.  Several possible technical reasons for this incomplete recovery were explored including possible cell loss, shifts in buffer pH, and nutritional deficien­cies of the incubation media, but none of these variables could account for the finding that formylpeptide receptor reexpression never exceeded 85% of the initial cell surface binding.  Nonetheless, no differences were seen in formylpeptide receptor reexpression in CT and CA serum pretreated cells.  Thus, the chemotactic defect in the CA serum pretreated group does not seem to result from altered or insufficient formylpeptide receptor reexpression.

A significant decrease in ³H-FMLP uptake was seen in rabbit PMN and in normal human PMN and mononuclear leukocytes when they were pretreated with CA serum.  A similar decrease was observed in PMN from CA patients.  This de­crease was not seen at early time points, but became apparent after 20 minutes of exposure to labeled FMLP.  Others have shown that Fc receptor-mediated phagocytosis of radiolabeled immune complexes was suppressed in human PMN and monocytes treated with fractions prepared from CA patient serum (7) and that phagocytosis was reduced in tumor patient monocytes (36).  Further, Naik et al. (37) found that fluid pinocytosis in PMN from patients with chronic myeloid leukemia was significantly reduced as compared to that of normal PMN.  Although phagocytosis, receptor-mediated endocytosis, and fluid-phase pinocyto­sis are distinctly different processes (38), it seems that CA serum may have an inhibitory effect on all three forms of endocytosis.  Accumulation of soluble ³H-FMLP at 37°C involves two concurrent cellular processes, receptor-mediated endocytosis (saturable uptake) and fluid-phase pinocytosis (non-saturable uptake).  The method employed here did not permit the contributions of each of these processes to be distinguished, but previous studies employing identical conditions have concluded that about 80% of formylpeptide uptake occurs by a receptor-mediated mechanism (29,39).  Moreover, it is evident from Figure 5 that uptake by rabbit PMN was saturable and thus primarily receptor-mediated.  Finally, preliminary experiments using ¹⁴C-polyethylene glycol, a marker for fluid phase pinocytosis, have shown no differences in uptake between CT and CA serum pretreated leukocytes after formylpeptide stimulation (unpublished data).  There is, however, an appar­ent contradiction between this finding (inhibition of receptor-mediated uptake) and the results of experiments on formylpeptide receptor down-regulation and reexpression which were not affected by CA serum pretreatment.  Although the latter are not altered in CA leukocytes under the conditions described, it is possi­ble that the effects of the CA serum may have dissipated during the lengthy incubations (60-180 min after completion of serum pretreatment) required to perform these experiments.  Inhibitory effects of serum may remain evident, however, in the former experiments since they required much shorter incubations (5-60 min) after serum treatment.  Further studies will be required to evaluate this possibility.

In general, formylpeptide receptor down-regulation and receptor reex­pression did not appear to be affected by pretreatment with cancer serum.  However, significant reductions in formylpeptide binding were observed in tumor patient mononuclear leukocytes and in cancer serum pretreated rabbit peritoneal PMN.  Reductions in chemoattractant uptake at 37°C were seen in cancer serum pre­treated rabbit PMN, human PMN, human mononuclear leukocytes and in tumor patient PMN.  These alterations in surface binding and internalization of formylpeptide may contribute to the depression of chemotaxis exhibited by these cells.  Further characterization of CA leukocytes and the effects of CA serum on leukocyte chemotaxis and fluid phase pinocytosis are in progress.

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FIGURES

Figure 1

Binding of ³H-FMLP to adherent, unstimulated leukocytes exposed to ³H-FMLP for 60 min at 4°C.

(A)  Serum pretreated normal leukocytes.  Adherent rabbit peritoneal PMN, human peripheral blood PMN, and human peripheral blood mononuclear leukocytes were pre­treated with CT or CA serum prior to exposure to ³H-FMLP.  (n=15, rabbit PMN; n=20, human PMN; n=12, human mononuclear leukocytes; mean + SD)  *  p<0.01  (B)  Patient peripheral blood leukocytes.  PMN and mononuclear leukocytes isolated from venous blood samples obtained from tumor patients or control subjects were exposed to ³H-FMLP at 4°C.  (n=3; mean + SD)  *  p<0.05 

Figure 2

Down-regulation of formylpeptide receptors in the presence of varying concen­trations of unlabeled FMLP by adherent rabbit peritoneal PMN pretreated with CT or CA serum.  Shown is a representative experiment (n=4; mean + SD). 

Figure 3

Time course of formylpeptide receptor down-regulation for adherent rabbit peritoneal PMN pretreated with CT or CA serum and incubated with 10 nM unla­beled FMLP.  Shown is a representative experiment (n=4; mean + SD).

Figure 4

 

Formylpeptide receptor recovery after down-regulation for adherent rabbit peritoneal PMN pretreated with CT or CA serum, exposed to 5 nM unlabeled FMLP, rinsed, and further incubated in buffer at 37°C.  Shown is the percent of cell surface receptors expressed as compared to cell samples not exposed to unla­beled FMLP (i.e., 100% receptor expression)  (n=3; mean + SD).

Figure 5

Uptake of ³H-FMLP (20 nM) at 37°C by adherent rabbit PMN pretreated with CT or CA serum.  Uptake of ³H-FMLP in the CA serum pretreated group is significantly reduced at 10 min (p<0.003), 20 min (p<0.001) and 40 min (p<0.015).  Shown is a representative experiment (n=8).

Figure 6

Uptake of ³H-FMLP at 37°C in the presence of 20 nM ³H-FMLP by adherent normal human leukocytes pretreated with CT or CA serum.  (A)  Uptake in the CA serum pretreated cells is significantly reduced at 40 min (p<0.01) and 60 min (p<0.04).  Shown is a representative experiment (n=6; mean + SD).  (B)  Uptake in CA serum pretreated mononuclear leukocytes is significantly reduced at 40 min (p<0.01) in the CA serum pretreated cells.  Shown is a representative experiment (n=5; mean + SD).  

Figure 7

Uptake of ³H-FMLP at 37°C in the presence of 20 mM ³H-FMLP by adherent PMN isolated from control subjects and tumor patients.  Uptake in the CA patient PMN is significantly reduced at 20 min (p<0.001), 40 min (p<0.001) and 60 min (p<0.002).  Shown is a representative experiment (n=3; mean + SD).

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