Modulation of Alveolar Macrophage Phagocytosis by Leukotrienes Is ...

Modulation of Alveolar Macrophage Phagocytosis by Leukotrienes Is Fc Receptor–Mediated and Protein Kinase C-Dependent Peter Mancuso and Marc Peters-Golden Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan We have previously established an important role for leukotrienes (LTs) in augmenting rat alveolar macrophage (AM) phagocytosis of Klebsiella pneumoniae opsonized with complete immune serum (IS), which contains the two well-known opsonins, immunoglobulin (Ig) G and complement (C). In this report, the specific opsonin requirements for LT modulation of AM phagocytosis and the dependence of this response on protein kinase (PK) C activity were investigated. Phagocytosis of K. pneumoniae opsonized with IS, non-immune serum, or heat-inactivated immune serum and of inert targets (IgG-opsonized fluorescent microspheres or C-opsonized sheep red blood cells) was examined. Inhibition of endogenous LT synthesis or action attenuated, whereas the addition of exogenous LTs augmented, phagocytosis only of targets opsonized with IgG. LTs had no effect on phagocytosis of C-opsonized or unopsonized targets. LTs did not affect adherence of IgG-opsonized targets, implying instead an enhancement of internalization. Macrophage internalization of phagocytic targets has previously been shown to require PKC activity. Pretreatment of AMs with the PKC inhibitors staurosporine or calphostin C, or with phorbol 12-myristate 13-acetate to deplete PKC, completely inhibited the ability of LTB4 and largely inhibited the ability of LTC4 to augment phagocytosis of IgG-opsonized microspheres. These results demonstrate that LT enhancement is confined to Fc receptor (FcR)-mediated phagocytosis. Moreover, PKC activation represents an important mechanism by which LTs promote FcR-mediated phagocytosis.

The resident alveolar macrophage (AM) patrols the alveolar epithelial surface of the lung and maintains sterility by phagocytosing and killing microorganisms (1). As a means of augmenting host defense, these resident cells secrete chemotactic substances such as leukotriene (LT) B4, C5a, and cytokines that recruit neutrophils from the peripheral circulation to the alveolar focus of infection (2). LTs are potent lipid mediators of inflammation derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. The enzyme 5-LO, in conjunction with its helper protein 5-LO activating protein, can oxygenate AA to form LTA4. This intermediate can be hydrolyzed to form the potent neutrophil chemoattractant LTB4, or conjugated with glutathione to form the cysteinyl-LTs (LTC4, LTD4, and LTE4), which elicit smooth-muscle contraction and microvascular permeability (3). (Received in original form May 19, 2000 and in revised form August 9, 2000) Address correspondence to: Dr. Marc Peters-Golden, Division of Pulmonary and Critical Care Medicine, 6301 MSRB III, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. E-mail: [email protected] Abbreviations: arachidonic acid, AA; alveolar macrophage, AM; bovine serum albumin, BSA; complement, C; C receptor, CR; Fc receptor, FcR; Hanks’ balanced salt solution, HBSS; heat-inactivated IS, HIS; immunoglobulin; Ig; immune serum, IS; 5-lipoxygenase, 5-LO; leukotriene, LT; nonimmune serum, NIS; protein kinase, PK; phorbol 12-myristate 13-acetate, PMA; standard error, SE; sheep red blood cell, SRBC. Am. J. Respir. Cell Mol. Biol. Vol. 23, pp. 727–733, 2000 Internet address: www.atsjournals.org

Although the roles of LTs in inflammatory diseases such as asthma are well established (3), their contribution to host defense is less well understood. An important role for LTs in host defense against bacterial pneumonia was suggested by our previous report that 5-LO knockout mice exhibited enhanced lethality and reduced bacterial clearance as compared with their wild-type counterparts after intratracheal administration of the gram-negative bacterium Klebsiella pneumoniae (4). This in vivo defect in bacterial clearance was associated with reduced phagocytosis and killing of K. pneumoniae in in vitro studies with AMs from 5-LO knockout mice, as compared with cells from wild-type mice. This phagocytic defect could be duplicated by treatment of AMs from normal animals with pharmacologic agents that inhibited LT synthesis (5). Phagocytosis in LT-deficient cells could be substantially restored by the addition of nanomolar concentrations of various 5-LO products (4, 5). Moreover, exogenous LTs had pharmacologic effects inasmuch as they enhanced phagocytosis of K. pneumoniae even in cells with intact LT synthetic capacity (5). Macrophage phagocytosis is optimal when particulate targets are coated with opsonins such as the complement (C) fragments, C3b and C3bi, or immunoglobulin (Ig) G, permitting interactions via cellular C and Fc receptors (CR and FcR), respectively (6). Both of these opsonins would be expected to be present in the alveolar space in the setting of bacterial pneumonia (2). Our previous studies demonstrating enhancement of in vitro phagocytosis by LTs were performed using bacteria opsonized with 1% immune serum (IS), which would likewise contain both C and anti–K. pneumoniae IgG. Binding of phagocytic targets to these opsonin receptors is known to activate a variety of signaling pathways, including increases in intracellular Ca2+, release and metabolism of AA, and activation of kinases, including protein tyrosine kinases of the Syk and Src families, phosphatidylinositol 3-kinase, the Ras/Raf-1 mitogen-activated protein (MAP) kinase pathway, and protein kinase (PK) C (6). The goals of the present study were to define (1) the opsonin receptors through which LTs enhance AM phagocytosis, and (2) the role of one of these signaling mechanisms, PKC activity, in mediating this ability of LTs to enhance phagocytosis. Our results indicate that LT modulation of phagocytosis is mediated exclusively by FcRs, and is largely dependent on active PKC.

Materials and Methods Cell Isolation and Culture Resident AMs were obtained from pathogen-free female Wistar rats weighing 125 to 150 g (Charles River Laboratories, Portage, MI) by lung lavage as previously described (7). A total of 96% of

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the cells obtained from lavage were identified as macrophages by a modified Wright–Giemsa stain (Diff-Quik; American Scientific Products, McGaw Park, IL) (7). After lavage fluid centrifugation at 4⬚C at 200 ⫻ g for 5 min, the pellet was resuspended in Hanks’ balanced salt solution (HBSS) and the cells were enumerated using a hemocytometer. The cells were centrifuged a second time and resuspended in RPMI 1640 (GIBCO, Grand Island, NY) to a final concentration of 5 ⫻ 105 cells/ml. The murine macrophage cell line RAW 264.7 was obtained from the American Tissue Type Culture Collection (ATCC) (Rockville, MD) and maintained in RPMI 1640 with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and penicillin G, streptomycin sulfate, and amphotericin B (GIBCO). For phagocytosis experiments, 1 ⫻ 105 RAW cells or AMs were adhered to glass eight-well Falcon culture slides (Becton-Dickinson, Franklin Lakes, NJ) for 1 h in RPMI 1640.

K. pneumoniae Preparation K. pneumoniae strain 43816, serotype 2, was obtained from ATCC and aliquots were grown in tryptic soy broth (Difco, Detroit, MI) for 18 h at 37⬚C. The concentration of bacteria in culture was determined spectrophotometrically (A600) (8).

Serum Preparation and Opsonization Specific IS was prepared as follows: K. pneumoniae were suspended in saline and 1% formalin for 24 h. Bacteria were then pelleted by centrifugation at 400 ⫻ g, washed three times with saline, and 0.1 ml (108 bacteria) of the bacterial suspension was administered to rats via an intraperitoneal (i.p.) injection. The formalin-killed bacterial suspension was then frozen at ⫺80⬚C. At 10 d after the primary inoculum, a second i.p. injection was given. At 7 d thereafter the rats were anesthetized with sodium pentobarbital and blood was collected and allowed to clot at room temperature for 4 h. The blood was then refrigerated overnight to allow the clot to retract. The clotted blood was centrifuged at 1,900 ⫻ g and the serum was aliquotted and stored at ⫺80⬚C for future experiments. This serum would contain specific anti–K. pneumoniae IgG as well as C. Non-immune serum (NIS) (containing C but lacking specific IgG) was prepared in a similar manner from rats that were not immunized. Heat-inactivated IS (HIS) (containing specific IgG but inactivated C) was prepared by heating IS to 60⬚C for 30 min in a water bath. Before each experiment, 20 ⫻ 108 K. pneumoniae were suspended in HBSS in a 5-ml snap-cap tube and opsonized by mixing the bacterial suspension with serum for 15 min at 37⬚C on a rotating platform. All experiments were performed on targets opsonized with sera at a concentration of 1%, in keeping with our previous protocol (5).

Phagocytosis of K. pneumoniae After the addition of 1 ⫻ 106 K. pneumoniae opsonized with 1% IS, 1% NIS, or 1% HIS, the AM cultures were mixed for 1 min with a plate shaker (Hoefer Instruments, San Francisco, CA). The AM cultures were then incubated for 30 min at 37⬚C. After the incubation period, the extracellular bacteria were removed by washing three times with HBSS. The monolayers containing bacteria were allowed to dry overnight and were then stained with Diff-Quik. For each slide, a standard pattern of high-powered fields was examined by light microscopy (⫻1,000) to enumerate 100 cells. By comparing the phagocytic index in the presence and absence of cytochalasin D (10), we have previously determined that 90% of the cell-associated bacteria using this method were actually internalized (5).

Phagocytosis of IgG-Opsonized Microspheres For experiments using IgG-opsonized microspheres, 1 ⫻ 107 microspheres were added to the AM monolayers. After a 30-min incubation at 37⬚C, the monolayers were washed with cold HBSS and incubated with rhodamine-tagged goat-antirabbit IgG for 30 min at 4⬚C to label the extracellular microspheres. To observe the fluorescent microspheres, the AM monolayers were examined (⫻1,000) using a Leitz Orthoplan microscope equipped with an argon laser fluorescent lamp with broad-band violet and rhodamine filters. The blue microspheres observed by using the broad band violet filter were determined to be internalized if they did not appear to be labeled with the rhodamine-tagged goat-antirabbit IgG when viewed with the rhodamine filter.

Pharmacologic Modulation of AMs In some experiments, cells were pretreated for 15 min with the 5-LO enzyme inhibitor zileuton (10 ␮M) (Abbott Laboratories, Chicago, IL), the cysteinyl-LT receptor antagonist, LY171883 (1 ␮M), or the LTB4 receptor antagonist LY292476 (1 ␮M) (both from Eli Lilly, Indianapolis, IN), each diluted in HBSS. The use of these pharmacologic agents at these doses was previously shown to inhibit LT synthesis and block the LT receptors (11, 12) and reduce phagocytosis of K. pneumoniae (5), respectively. AM monolayers were incubated for 30 min with PKC inhibitors staurosporine (Sigma) (50 nM) or calphostin C (Calbiochem, San Diego, CA) (750 nM). These have been reported to inhibit PKC (13, 14) and also inhibited phagocytosis by approximately 50% in preliminary dose–response experiments (data not shown). PKC depletion was accomplished by incubating cells with 1 ␮M phorbol 12myristate 13-acetate (PMA) (Sigma) for 18 h (15). Where appropriate, lipoxygenase metabolites (Cayman Chemical, Ann Arbor, MI) were added in HBSS to the AM monolayers 10 min before the addition of bacteria or microspheres.

Adherence of IgG-Opsonized Microspheres Preparation of IgG-Opsonized Microspheres Blue fluorescent carboxylate-modified microspheres (365/415 Fluospheres, 2 ␮m in diameter) were obtained from Molecular Probes (Eugene, OR) and were coated with IgG using the method of Cannon and Swanson (9). Briefly, microspheres were incubated with 10 mg/ml bovine serum albumin (BSA) (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS) for 1 h at 37⬚C. The BSA-coated microspheres were then washed three times with PBS and incubated with the IgG fraction of rabbit anti-BSA serum (1/500 dilution) (Sigma) for 30 min at 37⬚C with constant agitation using a rotating platform. The IgG-coated microsphere suspension was then cooled to 4⬚C for 10 min, washed three times with PBS, and resuspended in HBSS. Microspheres were enumerated using a hemocytometer and diluted to a final concentration of 20 ⫻ 107 microspheres/ml.

To assess adherence of IgG-opsonized microspheres to the surface of AMs, monolayers were first incubated with 5 ␮g/ml of cytochalasin D to inhibit internalization. At 30 min after the addition of IgG-opsonized microspheres, the total number of adherent microspheres was enumerated for 100 cells using the microscopic procedure described earlier for phagocytosis.

Phagocytosis of C-Opsonized Sheep Red Blood Cells CR-dependent AM phagocytosis was assessed using C-opsonized sheep red blood cells (SRBCs). C-opsonized SRBCs were prepared by the method of Wright and Silverstein (16). Briefly, 1 ⫻ 109 SRBCs were incubated with 50 ␮l of rabbit anti-SRBC IgM (Accurate Chemical, Westbury, NY) for 1 h. Complement fragments C3b and C3bi were fixed on the IgM-coated SRBCs after a 10-min incubation with 50 ␮l of C5-deficient human serum

Mancuso and Peters-Golden: Leukotriene Modulation of Phagocytosis

(Sigma). These were added in a ratio of 100:1 to monolayers of AMs and the murine macrophage cell line RAW 264.7 that had been pretreated with medium (control), 15 nM PMA, and/or 1 nM LTB4 for 10 min. After a 30-min incubation period, the monolayers were washed three times with HBSS. Water was then added to each well for 10 s, followed by 3⫻ saline to lyse the surface-bound SRBCs and to restore isotonic conditions. A phagocytic index was calculated as described earlier. Micrographs of AM and RAW cell phagocytosis of C-SRBCs were taken using a Leitz Orthoplan microscope (⫻1,000) equipped with a Diagnostics Spot-2 CCD digital camera.

Statistical Analysis A minimum of three replicate wells per condition were studied in each experiment and the number of individual experiments is indicated in the figure captions. Data are expressed as means ⫾ standard error (SE). Where appropriate, mean values were compared using a paired t test or a one-way analysis of variance (ANOVA) and the Student–Newman–Keuls test for mean separation. In all cases, a P value ⬍ 0.05 was considered significant.

Results Inhibition of Endogenous LT Synthesis Reduces AM Phagocytosis of K. pneumoniae Opsonized with IS or HIS, but Not NIS We previously identified a role for endogenous LTs in phagocytosis of K. pneumoniae opsonized with 1% IS (5). Because IS contains both C and IgG, in these experiments we sought to determine the specificity of LT modulation for phagocytosis mediated by these two opsonins. The baseline phagocytic indices were 48 ⫾ 9, 25 ⫾ 4, and 25 ⫾ 5, for bacteria opsonized with IS, NIS, or HIS only, respectively. As shown in Figure 1, preincubation of AMs with the 5-LO inhibitor zileuton reduced phagocytosis by about 40% only when bacteria were opsonized with IS or HIS. However, no reduction in phagocytosis by this inhibitor was ob-

Figure 1. Effect of endogenous LT synthesis inhibition on AM phagocytosis of K. pneumoniae opsonized with 1% IS, NIS, or HIS. AMs were pretreated in the absence (control) or presence of zileuton (10 ␮M) for 15 min before the addition of opsonized K. pneumoniae. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, using a paired t test.

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served when AMs were incubated with K. pneumoniae opsonized with NIS. Antagonism of the Plasma Membrane Receptors for LTB4 or Cysteinyl-LTs Reduces AM Phagocytosis of K. pneumoniae Opsonized with IS or HIS, but Not NIS As an alternative approach to examine the role of endogenous LTs in AM phagocytosis, we blocked the plasma membrane receptors for LTB4 and cysteinyl-LTs with receptor antagonists. Previously, we had shown that both of these classes of LT receptor antagonists decreased phagocytosis of K. pneumoniae opsonized with IS (5). As with zileuton, the inhibition of phagocytosis with antagonists of both the LTB4 and the cysteinyl-LT receptor (Figure 2) was observed only when K. pneumoniae was opsonized with sera containing IgG. Exogenous LTs Enhance AM Phagocytosis of K. pneumoniae Opsonized with IS or HIS, but Not NIS In the previous experiments, we established the importance of endogenous LTs as modulators of phagocytosis mediated by the opsonin IgG. Because we had previously demonstrated that phagocytosis could be enhanced by exogenously added LTs (5), we next explored the opsonin specificity of this response. AMs were incubated with exogenous LTs for 10 min before the addition of K. pneumoniae opsonized with either IS, NIS, or HIS. Exogenous LTB4 (Figure 3A) and LTC4 (Figure 3B), both at 1 nM, enhanced phagocytosis of K. pneumoniae opsonized with IS or HIS, but not NIS. Endogenous and Exogenous LTs Modulate AM Phagocytosis of IgG-Opsonized Microspheres but Have No Effect on C-Opsonized SRBCs As another approach to assess the opsonin specificity for LT modulation of AM phagocytosis, we used as phagocytic targets both (1) fluorescent microspheres coated with BSA– anti-BSA IgG complexes and (2) C-opsonized SRBCs. In

Figure 2. Effect of LT receptor antagonists on AM phagocytosis of K. pneumoniae opsonized with 1% IS, NIS, or HIS. AMs were pretreated with the LTB4 receptor antagonist LY292476 (A) or the cys-LT receptor antagonist LY171883 (B), both at 1 ␮M, for 15 min before the addition of opsonized K. pneumoniae. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, using a paired t test.

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Figure 3. Effect of exogenous LTs on AM phagocytosis of K. pneumoniae opsonized with 1% IS, NIS, or HIS. AMs were pretreated with LTB4 (A) or LTC4 (B) at 1 nM for 10 min before the addition of opsonized K. pneumoniae. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, using a paired t test.

contrast to serum as a source of opsonins, the latter methods ensure that the targets are coated with a single opsonin. They also permit discrimination of cell surface–associated targets from internalized targets. In the case of the microspheres, this was accomplished by the addition of a secondary antibody (rhodamine conjugated goat-antirabbit IgG) at the end of the phagocytosis interval which labels only cell surface–bound targets. Likewise, surface-bound C-opsonized SRBCs were removed by hypotonic lysis. AMs were able to phagocytose IgG-opsonized microspheres but not naked microspheres or microspheres coated only with BSA (data not shown). Under control conditions, the mean phagocytic index for the IgG-opsonized microspheres was 44 ⫾ 7. As seen with K. pneumoniae opsonized with IgG, inhibition of endogenous LT synthesis or antagonism of the plasma membrane LT receptors attenuated, whereas the administration of exogenous LTB4 or LTC4 enhanced (Figure 4), IgG-opsonized microsphere phagocytosis. Rat AMs neither bound nor phagocytosed C-opsonized SRBCs (Figure 5A) even after pretreatment with LTB4 or PMA. In contrast, cells of the macrophage line RAW 264.7 were able to accomplish both (Figure 5B). LTs Do Not Modulate Adherence of IgG-Opsonized Microspheres to AMs The effects of zileuton, LT receptor antagonists, and exogenous LTs on AM association with IgG-opsonized microspheres were examined in the presense of cytochalasin D, which allows binding but inhibits phagocytosis. Neither endogenous LT synthesis inhibition, LT receptor antagonists, nor exogenous LTs significantly influenced the binding of microspheres to the AMs (data not shown). These results in-

dicate that LTs modulate phagocytosis by enhancing the AM’s ability to internalize the IgG-opsonized microspheres. LT Modulation of Phagocytosis Is Dependent on Functional PKC As an initial step toward dissecting the molecular mechanisms by which LTs modulate FcR-dependent phagocytosis, we chose to focus on the role of PKC because it has been shown to be necessary for FcR-mediated phagocytosis (17) and LTs have been shown to stimulate PKC activity in phagocytic cells (18, 19). As expected, the PKC inhibitors calphostin C and staurosporine dose-dependently inhibited phagocytosis of IgG-opsonized microspheres and were used at concentrations approximating the concentrations that produced 50% inhibition of effect for phagocytosis, 750 nM and 50 nM, respectively (Figures 6A and 6B). Importantly, these PKC inhibitors completely abrogated the ability of exogenous LTB4 to augment phagocytosis, and largely abrogated the ability of LTC4 to augment phagocytosis (Figures 6A and 6B). Likewise, depletion of PKC, which was accomplished by an 18-h incubation with PMA, resulted in a complete abrogation of phagocytic enhancement by LTB4 and a substantial but incomplete abrogation of phagocytic enhancement by LTC4 (Figure 6C). To determine whether PKC inhibition was additive to 5-LO inhibition in attenuating phagocytosis, we examined the effects of zileuton with or without PKC inhibition. Interestingly, neither pharmacologic inhibition of PKC with staurosporine or calphostin C (Figures 7A and 7B) nor PKC depletion with PMA (Figure 7C), when accomplished in the presence of zileuton, reduced phagocytosis to levels lower than that seen with zileuton alone (Figure 7).

Figure 4. Effect of exogenous LTs, LT synthesis inhibition, and LT receptor antagonists on AM phagocytosis of IgG-coated microspheres. AMs were pretreated with LTB4 or LTC4 (1 nM) for 10 min (A), or zileuton (10 ␮M), LTB4 receptor antagonist LY292476, or cys-LT receptor antagonist LY171883, both at 1 ␮M (B), for 15 min before the addition of IgG-coated microspheres. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, using a paired t test.


Figure 5. Comparison of rat AM and RAW 264.7 cell phagocytosis of C-opsonized SRBCs. C-opsonized SRBCs were added in a ratio of 100:1 to monolayers of AMs (A) or the murine macrophage cell line, RAW 264.7 (B), which had been pretreated with LTB4 and 15 nM PMA for 10 min. After a 30-min incubation, cells were stained with Diff-Quik. Ingested SRBCs appear as round eosinophilic inclusions abundant within RAW 264.7 cells in B.

Discussion In our previous report (5), we demonstrated that the LTs play an important role in AM phagocytosis of K. pneumoniae. In those experiments, LTs augmented phagocytosis of K. pneumoniae opsonized with IS, which contained at least two important opsonins, IgG and C. The purpose of the present study was to determine the opsonic receptors through which LTs enhance AM phagocytosis and the role of PKC in mediating the ability of LTs to enhance phagocytosis. The results indicate that LT modulation of phagocytosis is mediated through FcR because LTs were found to enhance phagocytosis only with bacteria or microspheres opsonized with IgG. In addition, PKC activity is essential for the ability of LTs to augment phagocytosis because three different methods of reducing PKC activity blocked LT augmentation of phagocytosis. In these experiments, attenuation or augmentation of AM phagocytosis of K. pneumoniae was observed only when these bacteria were opsonized with serum containing IgG, suggesting that LT modulation of phagocytosis is FcR-dependent. However, this conclusion is limited by several ca-

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Figure 6. Effect of PKC inhibition or depletion on exogenous LT augmentation of AM phagocytosis of IgG-coated microspheres. AMs were pretreated with medium alone (control), staurosporine (50 nM) (A), or calphostin C (750 nM) (B) for 30 min, or with PMA for 18 h (C). Next, AMs were incubated with either LTB4 or LTC4 (1 nM) for 10 min before the addition of IgGcoated microspheres. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, by ANOVA.

veats. First, the IS used in these experiments is a complex mixture, which could contain opsonins other than IgG and C. Second, it is possible that carbohydrate moieties present on the surface of the bacteria would be recognized by the AMs independent of the opsonin receptors. Finally, the staining method used in these experiments did not definitively distinguish between internalized and surface-bound bacteria. These issues were addressed in the next series of experiments. Inert targets were coated with a single opsonin, IgG (microspheres) or C (SRBCs), with which to interact with the AM. This excluded the possibility that LTs modulated phagocytosis via some other opsonin that may have been present in rat serum or nonopsonically via a moiety that may be present on the surface of K. pneumoniae. In addition, these methods permitted a definitive determination of internalized targets. These experiments likewise revealed that effects of LTs on phagocytosis were limited to IgG-opsonized targets, providing more convincing evidence that LT modulation of phagocytosis requires engagement of the FcR. It was also notable that the rat AMs did not bind or phagocytose C-opsonized SRBCs. This result suggests that under the conditions employed in our experiments, AMs ex-

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Figure 7. Effect of PKC inhibiton or depletion, endogenous LT synthesis inhibition, or both on AM phagocytosis of IgG-coated microspheres. AMs were pretreated with medium alone (control), staurosporine (50 nM) for 30 min (A), calphostin C (750 nM) (B), or with PMA for 18 h (C), with or without zileuton (10 ␮M) for 15 min before the addition of IgG-coated microspheres. Data are expressed as mean phagocytic index (percentage of control) ⫾ SE (n ⫽ 3). *P ⬍ 0.05 with respect to the control, by ANOVA.

press very low levels of CR. Interestingly, two other reports indicate that rat AMs, as compared with AMs from other species or peritoneal macrophages, exhibited either absent or reduced phagocytosis of C-opsonized targets (20, 21). It is still possible, of course, that C-mediated phagocytosis might be subject to modulation by LTs in cells with this capability; this awaits further investigation. Moreover, these data indicate that phagocytosis of K. pneumoniae opsonized with NIS was in fact independent of C and therefore presumably dependent on other, as yet undefined, opsonins. We next addressed the possibility that LTs modulate FcR-dependent phagocytosis by increasing adherence of targets to AMs. This could result from increases in the number and/or binding affinity of FcR. However, neither the LT synthesis inhibitors, LT receptor antagonists, nor exogenous LTs influenced adherence per se. These results indicate that LTs modulate phagocytosis by enhancing the process of internalization rather than adherence. Internalization of phagocytic targets requires the transduction of signals from the FcR to the cytoskeletal machinery of the cell. To approach a possible mechanism whereby LTs might augment internalization, we considered signals known to be important for FcR-dependent phagocytosis

which have also been shown to be generated by phagocytic cells upon exposure to LTs. We chose to study PKC because it fulfills both these criteria; its activation is both required for IgG-dependent phagocytosis (17) and stimulated by LTs (18, 22). To test the hypothesis that LT augmentation of phagocytosis occurs via PKC activation, we used two strategies to block PKC action. First, AMs were pretreated with two chemically unrelated inhibitors of PKC catalysis, staurosporine and calphostin C. Second, PKC was depleted by incubating AMs with the agonist PMA for 18 h. This has been shown to cause activation of PKC and its subsequent proteolytic degradation (15). Consistent with previous observations (17), baseline phagocytosis of IgG-opsonized microspheres was reduced to about 50 to 60% of the control level after treatment with each pharmacologic inhibitor as well as by PKC depletion after prolonged incubation with PMA. We next explored the role of PKC in the enhancement of phagocytosis by exogenous LTs. Both LTB4 (18, 23) and cysteinyl-LTs (22) have been shown to activate PKC. Both PMA pretreatment as well as the two PKC inhibitors completely prevented the ability of LTB4 to augment phagocytosis of IgG-opsonized microspheres, suggesting that modulation of phagocytosis by LTB4 was entirely PKCdependent. In a similar manner, PKC inhibition/depletion largely, but not completely, abrogated the ability of LTC4 to augment phagocytosis. The fact that LTC4 modulation of phagocytosis was incompletely blocked by PKC inhibition suggests that the cysteinyl-LTs may activate other signal transduction pathways, such as the Mek-1/MAP kinase pathway (19) and mobilization of intracellular Ca2+ (24), which may play significant roles in phagocytosis (6, 25). Zileuton decreased AM phagocytosis of IgG-opsonized microspheres to about the same level as that observed with PKC inhibition. Interestingly, the combination of 5-LO inhibitors and PKC inhibition resulted in no further suppression of phagocytosis than that observed with either treatment alone. This suggests a possible interplay or redundancy between these two pathways. In this regard, not only can LTs activate PKC, but their synthesis in rat AMs is also dependent on PKC (26). In summary, we have shown that inhibition of LT synthesis or action attenuated phagocytosis only of IgG-opsonized targets, whereas the augmentation of rat AM phagocytosis with exogenous LTs was also limited to targets opsonized with IgG. Therefore, LT augmentation of phagocytosis is FcR-mediated. Inhibition of PKC activity blocked LT augmentation of phagocytosis, demonstrating a critical role for this signaling pathway in immune activation by these multifunctional lipid mediators. Acknowledgments: The authors thank Thomas G. Brock, Michael Coffey, and Joel A. Swanson for helpful discussion and Maria Diakonova for technical assistance. This work was supported by National Heart Lung, and Blood Institute Grant RO1-HL58897. Support for one author (P.M.) was provided by National Institutes of Health Training Grant T32 HL07749 and an American Lung Association of Michigan Research Fellowship Training Award.

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