Insoluble Immune Complex-stimulated Neutrophil ... - NCBI - NIH

American Journal of Pathology, Vol. 140, No. 3, March 1992 Copyright © American Association of Pathologists

Insoluble Immune Complex-stimulated Neutrophil Leukotriene B4 Production Is Dependent on FcyRII and FcoyRIII and Independent of Pertussis Toxin-sensitive Signal Transduction Pathways E. Crockett-Torabi,* C. W. Smith,t J. R. Kateley, R. Patterson,11 P. Tsai,* and J. C. Fantone* From the Department of Pathology,* University of Michigan Medical School, Ann Arbor, Michigan, the Department of Pediatrics, Section of Leukocyte Biology, Baylor College of Medicine, Houston, Texas, the Department of Immunology,* Sparrow Hospital, Lansing, Michigan, and the Department of Microbiology,II Michigan State University, East Lansing, Michigan

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc-yRII and Fc-yRIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis ofLTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc-yRII or Fc,yRiiI receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble IC& Howeverpertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc-yRII and FcyRIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc-yRII and Fc-yRIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils. (Am J Pathol 1992, 140:613-620)

One of the important mechanisms by which inflammatory reactions may be initiated is the formation or deposition of

immune complexes (ICs) in tissues.1' 2 Neutrophils have been shown to be an important effector cell in mediating IC-dependent tissue injury secondary to their ability to secrete reactive oxygen metabolites, lysosomal enzymes, and products of phospholipid metabolism (e.g., LTB4, platelet activating factor). The central role of arachidonic acid (AA) metabolites in hypersensitivity reactions and inflammation has been studied extensively.3 In particular, leukotriene B4 (LTB4) has been reported in substantial amounts in synovial fluids of patients with rheumatoid arthritis, spondylarthritis, and gout.4'5 It has been believed that the local generation of LTB4 by stimulated neutrophils functions to amplify the inflammatory response secondary to further stimulation and recruitment of neutrophils to sites of tissue injury. Neutrophils express several different Fc receptors (FcR) for immunoglobulin G (IgG), including FcoyRll (CDW32) and FcyRIII (CD16).67 Both FcyRll and EcyRIll bind ICs but not monomeric IgG molecules, and play an important role in immunophagocytosis both in vivo and in vitro.8'9 Although it has been suggested that human neutrophil FcyRIII serves primarily as a binding molecule to present ligand to FcyRII for subsequent transmembrane signaling,1014 recent studies from two different laboratories, Kimberly et al14 and our own,15 demonstrated that FcyRIII would directly induce signals and functional responses in human neutrophils. Although a number of studies have demonstrated the abilities of ICs and IgGcoated particles to induce LTB4 release in neutrophils in the presence of added excess amount of AA, the receptor-dependent mechanisms of signal transduction pathways by which ICs activate neutrophils to release LTB4 are not clearly defined and may be highly stimulus dependent. Therefore, in an attempt to more clearly define Supported by grants NIH-5-RO1-HL28737, NIH-1-R29-AI31436/7-01, and NIH-P50-DK39255 from the National Institutes of Health. Accepted for publication October 21, 1991. Address reprint requests to Dr. Elahe Crockett-Torabi, Department of Pathology/Box 0602, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, Ml 48109-0602.

613

614

Crockett-Torabi et al

AJP March 1992, Vol. 140, No. 3

the mechanisms by which ICs function to promote inflammatory cell activation and tissue injury, we investigated the mechanism(s) by which ICs induce LTB4 metabolism from endogenous AA in neutrophils and the role of Fc-y receptors in the signal transduction pathways. We present data indicating that FcyRII and FcyRIII can directly induce neutrophil LTB4 formation, and ICs in insoluble and soluble forms induce LTB4 release from endogenous AA in human neutrophils through distinct signal transduction pathways that are dependent on both Fc-yRII and FcyRIII.

Materials and Methods

Materials All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. Hank's blanced salt solution (HBSS) and pertussis toxin (PTx) (Islet-activating protein) were purchased from Gibco (Grand Island, NY) and List Biological Laboratories, Inc. (Campbell, CA), respectively. RPMI-1640 and fetal bovine serum were purchased from Whittakers Bioproducts, Inc. (Maryland).

Preparation of Immune Complexes Immune complexes were prepared with normal human serum albumin (HSA) and rabbit polyclonal IgG antibodies (Organon Teknika Corp., West Chester, PA) to HSA as previously described.15 Briefly, HSA was gel filtered on Waters PROTEIN-PAK300SW column (Waters, Millipore Corp., Milford, MA) using high performance liquid chromatography. Immune complexes were prepared by adding a constant amount of anti-HSA antibody to varying amounts of HSA. After incubation for 1 hour at 370C and overnight at 5cC, the complexes were then washed twice with cold phosphate-buffered saline (PBS) and resuspended in PBS. Insoluble ICs were prepared at equivalence, and soluble ICs were made at five times antigen excess.

Preparation of Neutrophils Human neutrophils were prepared from citrated venous blood obtained from healthy, drugfree donors. Standard isolation techniques employing Ficoll-Hypaque gradients were used followed by dextran sedimentation and hypotonic lysis to remove red cells.15'16

Monoclonal Antibodies Fab fragments of monoclonal antibodies (mAb) IV.3, specific for FcyRII were purchased from Medarex Inc. (West Lebanon, NH), and mAb 3G8 specific for FcyRIII was a gift of Dr. J. C. Unkeless (Mt. Sinai, New York, NY). Fab 3G8 was prepared by digestion with papain, and purified by passage over protein A-sepharose using an ImmunoPure Fab Preparation Kit by Pierce (Rockford, IL). No contamination with immunoglobulin heavy chains was detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) analysis. Monoclonal antibodies MY-7 and MY-9 directed against neutrophil surface molecules (i.e., CD13 and CD33) were purchased from Coulter Immunology (Hialeah, FL). The antineutrophil mAb, 4A5 was prepared as previously described,17 and was used as a binding control in this study.

Pertussis Toxin Treatment of Neutrophils Neutrophils (2 x 106 / ml) in RPMI-1 640 (containing 10% fetal bovine serum, 2 mM glutamine, and 5 mM EDTA), pH 7.4, were incubated at 370C with constant gentle agitation in the presence of 1 ,ug PTx/ml as described previously.15 After 2 hours, cells were washed twice with HBSS (no Ca2+ and Mg2+) and suspended in HBSS (no Mg2+).

Crosslinking of Fcy Receptors Neutrophils were incubated with Fab fragment of either lV.3 or 3G8 mAb, or mouse IgG (Organon Teknika Corp., West Chester, PA), at 40C for 60 minutes. Cells were washed with HBSS to remove the unbound mAbs and were incubated with pure F(ab')2 fragments of goat antimouse IgG (F[ab']2 fragment specific) (GAM) (Organon Teknica Corp., West Chester, PA) at 370C for 10 minutes allowing crosslinking of FcyR to occur.

Incubation of Neutrophils with Immune Complexes Neutrophils in the presence or absence of cytochalasin B (CB, 5 ,ug/ml, at 37°C for 5 min) were incubated with a specified concentration of A23187, GMLP or ICs at 370C in a water-bath with constant gentle agitation. At the end of incubation, cells were chilled on ice for 5 minutes to stop the reaction and centrifuged for 10 minutes at 2000 x G at 40C. Supernatants were immediately analyzed for their LTB4 contents by RIA, using the Amersham (Arling-

Insoluble Complex-stimulated Neutrophil LTB4 Production 615 AJP March 1992, Vol. 140, No. 3

ton Heights, IL) TRK.840 kit. The sensitivity of the assay for LTB4 is 0.0012 ng per assay tube. The antisera to LTB4 crossreacts 0.03% with LTC4 and LTD4, < 0.03% with PGE2, and other prostaglandins and AA, and 0.14% with 5(S), 12(S)-di HETE.

Superoxide Anion Production Assay Extracellular superoxide anion (0-2) production was measured by means of the superoxide dismutase (SOD)inhibitible reduction of ferricytochrome C as previously described.15 The amount of 0-2 produced is expressed as n moles 0-2/1 x 1 o6 cells per 30 minutes. All assays were performed in duplicate and data are expressed as mean + SEM of multiple experiments.

Results

Release of LTB4 from Human Neutrophils Induced by Immune Complexes In a preliminary series of experiments, neutrophils were incubated with various concentrations (5-50 ,ug antibody

protein) of soluble and insoluble ICs (37°C, 10 min) and the release of LTB4 was determined by RIA. The threshold dose for activation of lipoxygenase enzyme system by insoluble ICs was 5 ,ug Ab protein / 5 x 1 06 cells in 1 ml (Figure 1). Soluble ICs with an excess of antigen concentration at five times that of equivalence also induced LTB4 release, but approximately 10% of that induced by insoluble ICs. Saturation for IC-induced LTB4 release was achieved at a dose of 20-50 ,ug Ab protein for both soluble and insoluble complexes. To assess whether phagocytosis of insoluble ICs by neutrophils was required for initiation of LTB4 synthesis, cells (10 x 1 06/ml) were pretreated with cytochalasin B (CB) (5 ,uM) before exposure to ICs. Pretreatment of neutrophils with CB did not inhibit ICs-induced LTB4 release. In fact, CB enhanced ICs-induced LTB4 synthesis by 1.5- to threefold (data not shown). Cytochalasin B alone did not have any effect on the release of LTB4 from neutrophils. Human neutrophils (5 x 106 cells, in the presence of 5 jiM CB) incubated with insoluble ICs generated LTB4 in a time-dependent manner (Figure 2). Small, yet significant amounts of LTB4 were detectable only after 2-minute incubation with ICs, reaching a maximum at 20 minutes (3.7 ± 0.9 ng LTB4 for insol. ICs and 0.6 ± 0.3 ng LTB4 for sol. ICs). In contrast, the release of LTB4 by

2.0 -

*- soluble IC E30 Insoluble IC 1.6 -

~o

1.2 -

tn

0 x%*. 0.8

-

0.4 -

cro*,O", 0.0 Y~~~~~~~~~~~ 0 10

T

x

x

20

30

40

50

60

Immune complex conc. (,ug) Figure 1. Dose-response curvesfor immune-complexes-induced release ofLTB4 from human neutrophils. Neutrophils (5 x 10' cells, in the absence of CB) were incubatedfor 10 minutes at 37°C with different concentration of soluble and insoluble ICs (pg ofAb protein), and the amount of LTB4 was determined by RIA Representative figure of three separate experiments. Values represent mean + SEM of triplicate

samples.


616


1000

100

RIN

FMLP

*

A23187

*

Insoluble-ICs

-o-

Soluble ICs

(D 7-

x

u) D li

Figure 2. Effect of various stimuli on LTB4 synthesis in human neutrophils. Neutrophils (5 x 106 cells pretreated with 5 ug CB/ml, at 37°C, 5 min) were incubated with either5 uM A23187, 0.5 pMFMLP, or 25 ug insoluble ICs (Ab protein) for the times indicated at 37°C. After incubation the supernatants were collected and analyzed for LTB4. Data represents the mean + SEM for four separate ex-

Incubation Time (min.) FMLP and A23187-stimulated neutrophils occurred rapidly, reaching a maximum level within 2 minutes of incubation for FMLP (3.2 1.6 ng) and 5 minutes for A23187 (143 48 ng). Preincubation of neutrophils with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), completely inhibited IC-induced LTB4 release (data not shown). ±

±

Role of FcyRlI and FcyRlIl in LTB4 Release by IC-stimulated Neutrophils The role of specific FcyR in initiation of LTB4 synthesis by neutrophils, on stimulation with soluble and insoluble ICs was assessed using Fab fragments of mAb antibodies lV.3 and 3G8 to block ICs binding to Fc-yRII and FcyRIII, respectively. Pretreatment of neutrophils with either mAb markedly inhibited LTB4 release by IC-stimulated neutrophils, but did not inhibit FMLP-induced LTB4 release (Figure 3). The LTB4 release by IC-stimulated neutrophils was not inhibited after treatment of cells with the Fab fragments of mouse IgG or mAbs MY-7, MY-9, and 4A5 which served as controls. MY-7, MY-9, and 4A5 are antineutrophil antibodies that bind to specific surface molecules on human neutrophils.

Release of LTB4 Induced by Crosslinking of the FcyRII and Fc-yRIII Receptors on Neutrophils To determine whether the LTB4 generation by neutrophils stimulated with ICs was initiated by attachment of ICs to specific FcyR and crosslinking of these receptors,

periments.

examined the effect of direct crosslinking of FcyR on LTB4 release. Crosslinking of either Fc-yRII or Fc-yRIII induced significant LTB4 production by neutrophils (Table 1). When both receptors were simultaneously crosslinked, LTB4 release was significantly enhanced. In concert, the superoxide generated by neutrophils (from the same donors) upon crosslinking of either FcyRs was also potentiated when both Fc receptors were simultaneously crosslinked (Table 1). Pretreatment of control cells with Fab fragments of mouse IgG or mAbs MY-7, My-9 had no effect on LTB4 release or superoxide generation, indicating the responses to mAbs IV.3 and 3G8 were specific. When neutrophils were incubated with Fab fragments of 3G8 and IV.3 mAbs without crosslinking the FcyRII and FcyRIll molecules, no measurable amount of LTB4 or superoxide was detected (data not shown), indicating that the crosslinking of FcyR molecules triggered signals to activate the neutrophil.

we

Effect of Pertussis Toxin on LTB4 Formation in Neutrophils In a preliminary series of experiments, we previously'5 determined the optimal concentration of PTx by examining the inhibitory effect of different concentrations (101000 ng) of toxin and various time of incubation (370C, for 60-180 min) of toxin with cells, on functional responses of neutrophils in response to FMLP, PMA, and ICs. Similar to the studies by other investigators,18,19 we found that 1 jig PTx / 2 x 106 neutrophils in 1 ml was an optimal concentration that induced maximal inhibition (90-98%) of FMLP-induced superoxide generation in human neutrophils. The pertussis toxin treatment was not toxic to the

Insoluble Complex-stimulated Neutrophil LTB4 Production 617 AJP March 1992, Vol. 140, No. 3

*

*

*

**

*

100 '

*

IV.3+3G8

3G8 80

-

IVo3

El Ms.lgG

or/ Cont. mAb

6002

PCI

40 E-4

20

O

-

1.

---

INSOL.IC

SOLUIC

--I

FMLP

Figure 3. Effects of pretreatment with monoclonal antibodies on LTB4 release by human neutrophils stimulated with various stimuli. Neutrophils (1 x 10/mi) were preincubated with saturated concentrations of Fabfragments ofmAb; 3G8, IV3, or control mAbs MY-7, MY-9, 4A5, mouse IgG, and HBSSfor 1 hour at 4 °C Cells were then washed and incubated with CB (5 ug/ml)for 5 minutes at 370C and then added into the tubes containing specific stimuli and incubatedfurtherfor a period of time (i.e., 2 min for FMLP, 10 min for soluble ICs, and 20 min for insoluble ICs). LTB4 released by neutrophils into supernatants measured by RIA Inhibition ofLTB4 by mAbs and mouse IgG is expressed as the percent of controls (neutrophils preincubated in HBSS without mAbs or mouse IgG, and were then incubated further with various stimulants). Results are expressed as mean + SEM forfive separate experiments (different donors). *The response was significantly different from controls at P = 0.001 by the analysis of variance (ANOVA) test.

cells as judged by trypan blue exclusion. Pretreatment of cells with PTx (1 ,ug PTx/2 x 106 cells, 37°C, 120 min) resulted in a significant inhibition of LTB4 release in neutrophils stimulated with either soluble ICs or FMLP (Table 2). In contrast, PTx treatment had minimal effect on insoluble IC-induced LTB4 release. LTB4 formation in response to A23187 stimulation was also not significantly inhibited by PTx treatment. Since there was a great difference in the magnitude of LTB4 production by soluble and insoluble ICs, we examined pertussis toxin sensitivity of ICs at concentrations of these two stimuli which stimulated identical production of LTB4. These studies confirm the inhibition of soluble ICs stimulation by pertussis toxin, and the lack of effect on insoluble ICs stimulation of neutrophil LTB4 production (Figure 4). In addition, superoxide generated by stimulated neutrophils upon simultaneous crosslinking of both FcyRs was sensitive to PTx inhibition (data not shown). To ensure that the toxin treatment was not affecting the expression of Fc-yR on neutrophil surface, FcyRII and FcyRII receptor expression on neutrophils with and without PTx treatment was quan-

titated. Flow cytometry analysis showed no apparent changes in the cell surface expression of either FcyRII or FcyRlII receptors on neutrophils after PTx treatment, indicating that the receptors were present and available on the cell surface for ligand binding (data not shown).

Discussion The data presented in this study demonstrate that immune complexes via interaction with Fc-yRII and FcyRIII receptors stimulate peripheral blood neutrophils to release LTB4 in the absence of exogenous A. Insoluble ICs were more potent stimulators when compared with soluble complexes. Immune complexes in soluble form, induce activation of neutrophils through biochemical pathways that are sensitive to inhibition by PTx. This effect is similar to that observed with PTx inhibition of neutrophil stimulation by chemotactic peptides such as FMLP and suggests that similar signal transduction pathways (i.e., G-protein and phospholipase C dependent)

618



Table 1. Effects of Fc-y Receptor Crosslinking on LTB4 Release and 0-2 Generation in Human Neutrophils 0-2

production (n moles/ First incubation

Second incubation

HBSS HBSS IV.3 (Fab) 3G8 (Fab) 3G8 + IV.3 (Fab) Mouse IgG (Fab)

HBSS FMLP GAM [F(ab')2] GAM [F(ab')2] GAM [F(ab')2] GAM [F(ab')2]

1 x 106

cells) 0.3 14.2 2.4 4.9 10.2

+ 0.1 ± 0.8* ± 0.3* 0.3* ± 2.0*

0.2 ± 0.1

LTB4 release (ng/5 x 106 cells)