Sepsis: Effects on Polymorphonuclear Fc Receptor-Mediated Phagocytosis
H. HANK SIMMS, M.D., Department
of Surgery, Rhode Island Hospital,
593 Eddy Street, Providence,
Rhode Island 02903
Submitted for publication March 30, 1989
studies used serum-opsonized particles as phagocytic targets. Multiple types of independently functioning, specific receptors for individual ligands exist on the PMN surface. Therefore, the different responses seen may have been secondary to multiple receptor-ligand interactions with these serum-opsonized particles. Consequently, phagocytic function may be better examined using target particles which bind only to individual receptors. In a recent report, adherent PMN from normal, healthy individuals were shown not to express full phagocytic potential for sheep erythrocytes opsonized with IgG (EIgG). However, phagocytic activity was markedly enhanced by sodium azide or catalase, which are inhibitors of various steps in the oxidative burst [ 71. This observation was not limited to phagocytosis of sheep erythrocytes as this enhancement of phagocytosis by sodium azide was also shown with serum-opsonized Cundidu. PMN from chronic granulomatous disease (CGD) patients and from one patient with myeloperoxidase deficiency showed markedly enhanced phagocytosis in comparison to normal PMN , These findings indicated that products of oxidative respiration regulate the phagocytic uptake of IgG-sensitized targets in normal PMN and suggested that the degree of ingestion of targets is under physiologic control. Patients with acute bacterial infections have demonstrated marked enhancement of PMN phagocytosis despite the absence of inhibition of the respiratory burst (upregulation). This increase in function returned to normal concomitant with clinical improvement . The natural history of PMN function during untreated and treated sepsis can be studied only in animal models. Animal models are useful only as they simulate phenomena recognized in humans. This paper reports the results of studies designed to assess the state of activation of swine PMN with regard to Fc receptor-mediated phagocytosis following untreated and treated intraabdominal sepsis. The target particles consisted of either sheep erythrocytes (E) or erythrocytes coated with known amounts of IgG (ElgG). Experiments were conducted to assess (1) the relationship between swine oxidative metabolism and PMN phagocytosis and (2) the effects of
In vitro studies have shown that phagocytic cells are capable of undergoing activation in response to inflammatory signals and that the activation process is quite complex. A relationship between polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis and oxidative metabolism has been seen in humans. We have sequentially examined circulating polymorphonuclear leukocytes (PMNs) from a total of 13 postoperative swine with either no sepsis, untreated intraabdominal sepsis, or treated intraabdominal sepsis to determine phagocytic activity over 8 postoperative days (POD). Products of the oxidative burst (i.e., myeloperoxidase) reduced the phagocytic activity of nonseptic swine PMN. Phagocytic activity was augmented by inhibiting the nonseptic swine oxidative burst with 10 mM sodium aside (an inhibitor of myeloperoxidase). In swine with untreated intraabdominal sepsis, PMN Fc receptor-mediated phagocytosis exhibited a biphasic response. An initial (between POD1 and POD4) increase in PMN function was followed by a subsequent (between POD4 and PODS) decrease in PMN function. Partial preservation of phagocytic capability was seen when swine were reexplored on POD4 and had their intraabdominal sepsis treated. These results indicate that (1) as in humans, nonseptic swine PMN Fc receptor-mediated phagocytosis is augmented by inhibition of the PMN respiratory burst; (2) untreated intraabdominal sepsis produces an initial increase and subsequent decrease in PMN Fc receptor-mediated phagocytosis; (3) early treatment of intraabdominal sepsis results in partial restoration of PMN Fc receptor-mediated phago0 1990 Academic Press, Ino. cytosis.
Phagocytosis by polymorphonuclear leukocytes (PMN) constitutes a critical mechanism of host defense against bacterial infection. Many in vitro studies have detailed phagocytic changes of normal PMN responding to inflammatory and infectious stimuli [ 1,2]. Increased [3,4], normal, and decreased [5,6] phagocytic capabilities have been reported in patients with infection. In general, these
Copyright 0 1990 by AcademicPress, Inc. rights of reproductionin any form reserved.
untreated and treated intraabdominal sepsis on PMN Fc receptor-mediated phagocytosis. Studies in 13 postoperative swine (both nonseptic animals and animals with intraabdominal sepsis) revealed that, as in humans, nonseptic swine PMN Fc receptormediated phagocytosis is enhanced with inhibition of the PMN respiratory burst. Second, PMN from animals with untreated intraabdominal sepsis demonstrated a biphasic response in their function with an initial increase, between postoperative day (POD) 1 and POD4, and a subsequent decrease, between POD4 and POD8, of PMN function. Finally, surgical intervention on POD4 resulted in a partial preservation of PMN phagocytic function when these animals were restudied 4 days later. MATERIALS
Buffers and Media Veronal-buffered saline (VBS), low-ionic-strength buffer containing 60% dextrose in VBS (DVBS), and VBS containing 10 mM ethylenediaminetetraacetate (EDTAVBS) were prepared as previously described [9, lo]. Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS2-) and RPMI-1640 Laboratories, Grand Island, New York. Reagents Reagents were obtained from the following sources: sodium azide, Fisher Scientific Company, Fairlawn, New Jersey; lymphocyte separation medium, Litton Bionetics, Kensington, Maryland; Dextran T500, Pharmacia Fine Chemicals, Uppsala, Sweden. Cellular Intermediates The IgG fraction of a rabbit anti-sheep RBC antibody was purchased from Cappel Laboratories, West Chester, Pennsylvania. Sheep erythrocytes (E) were washed with EDTA-VBS and VBS and resuspended to 1 X 10’ cells/ ml in VBS. Varying dilutions of the IgG antibody were added to E followed by a 15min incubation at 30°C and three washes in VBS. The cells were resuspended to 1.5 X lO’/ml in DVBS for phagocytosis assays. Previous experiments using radiolabeled IgG antibody demonstrated that cells sensitized with a 1:500 dilution of this antibody have approximately 4580 molecules of IgG per cell. Preparation
PMN were separated from venous blood samples by Ficoll-Hypaque density centrifugation and dextran sedimentation [ll]. The erythrocytes were removed by two hypotonic saline lysis steps. The PMN were washed two additional times and resuspended in RPM1 to 5 X 105/ ml for phagocytosis assays.
VOL. 49, NO. 1, JULY
Animal Preparation Female swine weighing 20-25 kg were anesthetized with ketamineim and thiamylal sodium (Surital) iv. Under general anesthesia and endotracheal intubation, the right common femoral artery and vein were catheterized with a Swan-Ganz catheter (Edward Laboratories, Santa Ana CA) and 7-Fr catheter, respectively. Then, either animals underwent sham laparotomy or intraabdominal sepsis was induced using a standardized cecal ligation and incision technique. Previous autopsy studies revealed that all animals in the cecal ligation and incision group had an intraabdominal abscess with generalized peritonitis by POD8. Animals received 500 ml of normal saline during the surgical procedure and were then allowed to eat and drink ad libitum until they were sacrificed on POD8. To assess the effect of surgical intervention on PMN function, a smaller group (n = 3) of animals underwent drainage of the abscess and establishment of a diverting cecostomy on POD4 and were restudied 4 days later (treated POD8). In contradistinction to untreated animals, these animals had negative blood cultures obtained by cardiac puncture just prior to autopsy. Phagocytosis Assays Immediately after line placement, but before surgery and on PODl, POD4, and POD8, PMN were sampled from the PA port of the Swan-Ganz catheter. In these assays, 250 ~1 of PMN at 5 X lO’/ml were plated in each chamber of slide-mounted, eight-chamber tissue culture plates (Lab-Tee, Naperville, IL) and allowed to adhere for 45 min at 37°C in a 5% CO2 incubator. Either E or EIgG (100 ~1) was added to the adherent PMN and the plates were centrifuged at 5Og for 5 min at 4°C. Phagocytosis was stopped after 40 min at 37“C by immediately adding 0.2% NaCl to remove external RBC. After two exposures to 0.2% NaCl, the cells were fixed with glutaraldehyde and stained with Glemsa. To assess the effects of sodium azide (an inhibitor of myeloperoxidase) on phagocytosis, an aliquot of PMN at 5 X 105/ml was exposed to 10 mM sodium azide for 15 min at 30°C prior to adherence in the culture plates. Previous investigations have shown that at this ooncentration, the primary effect of sodium azide is inhibition of myeloperoxidase . PMN which did not receive sodium azide had added buffer and were also incubated at 30°C for 15 min. The percentage of PMN ingesting one or more red cells was determined microscopically (100X; oil immersion) and the number of erythrocytes ingested per 100 PMN calculated (phagocytic index). Endotoxin
At the time that blood was withdrawn for PMN preparation, serum samples were drawn for endotoxin measurement utilizing the chromogenic limulus lysate assay
Percentage Phagocytosis of EIgG Untreated Intraabdominal
in Animals Sepsis”
l/500 l/1000 l/2000
23.80 + 9.99 15.20 f 7.49 6.80 k 3.03
41.60 + 8.96* 33.20 5~9.65* 12.80 + 4.97*
19.20 f 3.49** 7.60 + 2.30** 2.80 + 2.28**
“r&=5. * P < 0.005, POD1 vs POD4 at each IgG dilution. ** P < 0.005, POD4 vs POD8 at each IgG dilution.
IgG Dilution FIG. 1. Phagocytosis of EIgG by normal swine immediately after sham laparotomy: PMN at 5 X 105/ml either with or without 10 m&f sodium azide in RPM1 were adhered in Lab-Tee chambers for 40 min at 37°C. 100 ~1 of EIgG at 1.5 X lOs/ml was added to each chamber and phagocytosis was assayed after 40 min at 37°C. At each dilution of IgG, phagocytosis of EIgG was enhanced by the addition of sodium azide.
in both treated and untreated animals. The limit of endotoxin detection in the assay was 10 pg/ml (Kit QCL1000, MA-Bioproducts, Walkersville, MD). Statistical
All experiments were analyzed using the unpaired t test. Results reported are means -+ SD. Values with P < 0.05 were considered statistically significant. RESULTS Effect of Sodium Azide on Phagocytosis Phagocytosis assays were performed to determine if the PMN of normal swine would ingest IgG-coated particles under conditions in which there was only minimal ingestion by human PMN. The results after 40 min of phagocytosis are shown in Figs. 1 and 2. All experiments were performed immediately after the sham laparotomy.
In the absence of sodium azide, the percentages of swine PMN ingesting EIgG at l/500, l/1000 and l/2000 dilutions of IgG were 21.14 +_ 11.97, 13.42 f 7.30, and 5.71 f 3.81, respectively. The percentages of swine PMN ingesting EIgG in the presence of sodium azide at equivalent dilutions of IgG were 46.57 ? 11.68, 34.85 + 12.04, and 21.85 f 11.86, respectively. The phagocytic indices for ingestion of EIgG with or without azide at 1/500,1/1000, and l/2000 dilutions of IgG were 131.00 f 24.11 vs 53.00 f 16.47, 79.57 t- 23.24 vs 23.00 f 13.82, and 31.00 +- 13.67 vs 9.57 -+ 7.89, respectively. Phagocytosis of E alone was not seen. (N = 5; P < 0.005 comparing phagocytosis and phagocytic index with and without sodium azide at each IgG dilution.) Effects of Intraabdominal
Sepsis on Phagocytosis
Phagocytosis assays (in the absence of sodium azide) were performed on PODl, POD4, and POD8. Significant differences in phagocytosis were not seen between POD0 (after line placement and before laporotomy) and PODl. PMN Fc receptor-mediated phagocytosis exhibited a biphasic response. The percentage phagocytosis increased significantly between POD1 and POD4 and then fell significantly between POD4 and POD8 (Table 1). Similar changes were seen in the phagocytic index (Table 2). Effects of Surgical Intervention
The results of surgical intervention on PMN Fc receptor-mediated phagocytosis are shown in Figs. 3 and 4. At H •a
Indices of EIgG in Animals Intraabdominal Sepsis
l/500 l/1000 l/2000
45.40 f 10.73 21.20 k 1.45 12.60 + 4.85
102.00 k 17.02* 61.20 t 9.89* 29.00 + 6.69**
35.80 f 3.49* 13.60 k 6.16’ 5.20 + 2.66*
IgG DILUTION FIG. 2.
to data presented in Pi.
* P < 0.05, POD1 vs POD4 at l/500 and l/1000 dilutions; POD8 at l/500, l/1000, and l/2000 dilutions. ** P = 0.058, POD1 YS POD4 at l/2000 dilution.
VOL. 49, NO. 1, JULY
0 1 I500
IgG Dilution FIG. 3. Phagocytosia of EIgG by swine with both treated and untreated intraabdominal sepsis. Swine were reexplored on POD4 and underwent drainage of their abdominal abscess with creation of a diverting cecostomy. Treatment of intraabdominal sepsis resulted in increased phagocytosis at l/500 and l/1000 dilutions of IgG.
IgG dilutions of l/500, l/1000, and l/2000, the percentages of untreated swine PMN ingesting EIgG were 19.20 + 3.49, 7.60 + 2.30, and 2.80 f 2.28, respectively. The percentages of treated swine PMN ingesting EIgG at equivalent IgG dilutions were 64.75 _+2.63, 27.75 -+ 2.75, and 3.25 + 1.71, respectively. (N = 5 untreated and 3 treated animals; P < 0.006 for l/500 and l/1000 dilutions, respectively; P = ns at l/2000 dilution.) At IgG dilutions of l/500, l/1000, and l/2000 the phagocytic indices for untreated swine ingesting EIgG were 35.80 f 3.49, 13.60 + 6.16, and 5.20 + 2.66, respectively. The phagocytic indices for treated animals at equivalent IgG dilutions were 179.04 f 22.63,59.37 t- 13.22, and 5.00 f 3.70, respectively. (P < 0.01 for l/500 and l/1000 dilutions; P = NS at l/ 2000 dilution.)
Serum Endotoxin Measurements To determine if the changes in PMN function seen were due to concomitant changes in circulating endotoxin levels, serum samples were drawn from the PA port of the Swan-Ganz catheter at the same time PMN preparations were done. The results for both treated and untreated animals are shown in Table 3. Mean endotoxin levels peaked on POD8 at 2.78 -+ 1.03 rig/ml in untreated animals; mean endotoxin level peaked on POD4 at 1.901 +_ 0.326 rig/ml and dropped to 0.521 + 0.083 rig/ml in treated animals.
to data presented in Fig. 3.
cently been investigated. In the absence of sodium azide or other oxygen radical scavengers, PMN from healthy individuals show minimal phagocytosis of erythrocyte targets even when these targets are coated with subagglutinating amounts of IgG (-18,000 IgG/cell) . Patients with chronic granulomatous disease and those patients whose PMN are myeloperoxidase deficient have increased levels of Fc receptor-mediated phagocytosis [7, 121. In addition, platelet-derived growth factor, which depresses the normal oxidative burst, enhances phagocytosis . In this report, we investigated the effects of inhibition of the nonseptic swine oxidative burst to determine if nonseptic swine PMN would exhibit characteristics similar to those seen in humans. As shown in Figs. 1 and 2, inhibition of the swine oxidative burst using 10 mM sodium azide (to inactivate myeloperoxidase) enhanced PMN Fc receptor-mediated phagocytosis. This effect was seen at various dilutions of IgG antibody representing -4500 to 18,000 molecules IgG per red blood cell. The importance of studying PMN function in a receptor-specific fashion has recently been reported by several authors. Tosi et al. have demonstrated that the PMN Fc receptor (FcRIII, it& = 50-70 kDa) is elastase sensitive,
TABLE Mean Endotoxin Endotorin
3 Levels’ (rig/ml)
0.173 k 0.021 0.155 a 0.019
0.736 f 0.112* 0.745 f 0.119
1.890 + 0.321 1.901 f 0.326”
2.78 + 1.03’ 0.521 + 0.063**’
Phagocytosis by various immunologically active cells constitutes a critical part of the immune system. Polymorphonuclear leukocytes represent an important component of this system and their role in both healthy persons and patients with acute bacterial infections has re-
“n=5. * P < 0.05, POD8 VBpresurgery for untreated and treated animals. ** P < 0.05, POD8 vs POD1 for untreated animals. *** P < 0.05, POD4 vs POD8 in treated animals; POD8 in treated v8 untreated
whereas the PMN Fc receptor (FcRII, A& = 40 kDa) is not elastase sensitive. More importantly, superoxide anion production is reduced significantly when the functional epitope on FcRII but not FcRIII is bound . Fc receptor-mediated phagocytosis in patients with acute bacterial infections is initially increased and then falls to normal levels with appropriate treatment . In the swine with untreated intraabdominal sepsis, there was also a marked increase in Fc receptor-mediated phagocytosis. This was demonstrated by the fact that in the absence of inhibition of the oxidative burst, phagocytosis of EIgG increased significantly between POD1 and POD4 (upregulation) (Tables 1 and 2). In humans, PMN Fc receptor-mediated phagocytosis falls to normal levels concomitant with an improvement in the patients’ clinical condition . In animals with untreated intraabdominal sepsis, PMN Fc receptor-mediated phagocytosis falls to levels at or below that seen on POD1 (Tables 1 and 2). These results suggest that 8 days of untreated intraabdominal sepsis produces a suppression of PMN Fc receptor-mediated phagocytosis. Specifically, it would appear, therefore, that in early intraabdominal sepsis (POD1 to POD4) the stimulatory effects of sepsis overcome the inhibitory effects of respiratory burst products on PMN Fc receptor-mediated phagocytosis. In contradistinction to prolonged untreated intraabdominal sepsis (POD4 to POD8), products of the respiratory burst downregulate swine PMN Fc receptor-mediated phagocytosis; the exact mechanism for this is currently being investigated. In a smaller group of animals, surgical therapy on POD4 resulted in preservation of PMN function compared to animals without surgical intervention (Figs. 3 and 4). The beneficial effects of surgery were seen at IgG dilutions that led to maximal opsonization of target particles. These results suggest that with less than maximal binding of the Fc receptor, binding of the complement opsonic receptors (CRl, CR3) may be necessary for preservation of immune function after appropriate surgical intervention. Nonetheless, coating of sheep erythrocytes with complement alone (C3b or C3bi) does not lead to appreciable amounts of phagocytosis either in humans  or in our animal model of sepsis (manuscript in preparation). Therefore, ligation of both Fc and complement receptors appears necessary for optimal phagocytosis of target particles. During prolonged untreated sepsis, if respiratory burst products are able to adversely affect PMN Fc receptors (number, distribution, or affinity for ligand) then one would predict an ultimate reduction in phagocytic capability. This hypothesis is currently being tested. Endotoxin levels gradually increased in animals with untreated intraabdominal sepsis, but did not correlate temporally with the biphasic response seen in PMN function. Endotoxin levels dropped between POD4 and POD8 in treated animals while phagocytic capabilities improved.
This implies that the improvement in phagocytosis was not due to changes in endotoxin levels as increased levels of phagocytosis have generally been associated with increasing (not decreasing) levels of endotoxin. These results suggest that (1) as in humans, nonseptic swine PMN Fc receptor-mediated phagocytosis is augmented by inhibition of the PMN respiratory burst; (2) untreated intraabdominal sepsis produces an initial increase and subsequent decrease of PMN Fc receptor-mediated phagocytosis; (3) early treatment of intraabdominal sepsis results in partial restoration of PMN Fc receptormediated phagocytosis. In conclusion, these results suggest that intraabdominal sepsis induces an initial upregulation of PMN phagocytosis which presumably would be of benefit for the host. Of note, however, is the fact that in spite of ongoing sepsis PMN phagocytosis eventually falls, and, even though surgical intervention improves PMN phagocytosis, this requires maximal opsonization of the target particles. Should the septic host be unable to provide this degree of opsonization of target particles (e.g., bacteria) then phagocytosis of target particles would presumably be impaired. REFERENCES 1.
Smith, C. W., Hollus, C., Patrick, R. A., and Hossett, C. Mobility and adhesiveness in human neutrophils. Effects of chemotactic factors. J. Clin. Znuest. 63: 221, 1979.
W., Seligmann, B., and Gallin, J. I. Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine and increases complement C3bi receptor expression. J. Clin. Znuest. 77: 925, 1986.
3. Barbous, A. G., Alfred, C. D., Solberg, C. O., and Hill, H. R. Chemiluminescence by PMNs from patients with active bacterial fections. J. Infect. Dis. 141: 14, 1980.
4. Krukowski, function 1983.
Z. H., and Smith, G. A unifying concept of PMN in acute bacterial infections. World J. Surg. 7(3): 424,
5. Solberg, C. O., and Hellum, K. B. Neutrophil in bacterial
Lancet 2: 727, 1972.
6. Van Dijk, W. C., Vergrugh, H. S., Van der Tol, M. E., Peters, R., Peterson, P. K., Quie, P. G., and Verhoef, J. Interactions of phagocytic and bacterial cells in patients with bacteremia caused by gramnegative rods. J. Infect. Dis. 141: 441, 1980.
7. Gaither, T. A., Medley, S. R., Gallin, J. I., and Frank, M. M. Studies of phagocytosis
11(2):211,1987. 8. Simms, H. H., Frank, M. M., Quinn, S. H., Holland, S., and Gaither, T. A. Studies of phagocytosis in patients fections. J. Clin. Invest. 83: 252, 1987.
9. Mayer, M. M. Complement
with acute bacterial
and complement Kabat (Ed.), Experimental Immunochemistry. Charles C. Thomas, 1961. Pp. 151-209.
fixation. In E. A. Springfield, IL:
10. Rapp, J. J., and Borsos, T. Mokcukzr Basis of Complement Actions. New York: Appleton-Century-Croft,
11. Boyum, A. Isolation
1970. Pp. 75-134.
of mononuclear cells and granulocytes human blood. J. Clin. Lab. Invest. (Suppl.) 21: 77, 1968.
Gresham, H. D., McGarr, J. A., Shackelford, P. G., and Brown, E. J. Studies on the molecular mechanisms of human Fc receptormediated phagocytosis. J. Clin. Znuest. 82: 1192, 1988. Wilson, E., Costa, S. M., Gooding, L. R., Platelet-derived growth factor stimulates blocks agonist-induced activation of the burst: A possible cellular mechanism oxygen radical damage. Proc. Natl. Acad. 1987.
and Lambeth, J. D. phagocytosis and neutrophil oxidative to protect against Sci. USA 84: 2213,
Tosi, M. F., and Berger, M. Functional differences between the 40 kDa and 50 to 70 kDa IgG Fc Receptors on human neutrophils
49, NO. 1, JULY
revealed by elastase treatment and antireceptor antibodies. J. Immund 141: 2097,1987. Tennenberg, S. D., Zemlan, F. P., and Solomkin, J. S. 1988. Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. J. Zmmund. 141: 3937, 1988. Czop, J. K., Puglisi, A. V., Miorandi, D. Z., and Austen, K. F. Perturbation of fl-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B, production. J. Zmmunol.
Klebanoff, S. J., Vadas, M. A., Harlan, J. M., Sparks, L. H., Gamble, J. R., Agosti, J. M., and Waltersdorph, A. M. Stimulation of neutrophils by tumor necrosis factor. J. Immunol. 136(11): 4220,1986.