Amelioration of Lupus-Like Autoimmune Disease in NZB/W F1 Mice After

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8563-8568, August 1996 Immunology

Amelioration of lupus-like autoimmune disease in NZB/W F1 mice after treatment with a blocking monoclonal antibody specific for complement component C5 (immune complex/glomerulonephritis/inflammation) YI WANG*t, QILE Hu*, JOSEPH A. MADRIt, SCOrr A. ROLLINS*, AMY CHODERA*, AND Louis A. MATIS*t *Immunobiology Program, Alexion Pharmaceuticals, Inc., New Haven, CT 06511; and tDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510-8023 Communicated by David W. Talmage, Webb-Waring Institute for Biomedical Research, Denver, CO, May 6, 1996 (received for review February 17, 1996)

ABSTRACT New Zealand black x New Zealand white confirmed a role for terminal complement activation in the (NZB/W) F1 mice spontaneously develop an autoimmune progression of renal disease (5-7). syndrome with notable similarities to human systemic lupus We have been studying the contribution of activated termi- erythematosus. Female NZB/W F1 mice produce high titers of nal complement components to inflammatory disease pro- antinuclear antibodies and invariably succumb to severe cesses, using monoclonal antibodies (mAbs) specific for CS (8, glomerulonephritis by 12 months of age. Although the devel- 9). Functionally, these mAbs inhibit the cleavage of CS, thus opment of the immune-complex nephritis is accompanied by blocking the generation of the potent proinflammatory mol- abundant local and systemic complement activation, the role ecules C5a and CSb-9 (terminal complement complex), but not of proinflammatory complement components in disease pro- preventing the formation of C3b, which subserves critical gression has not been established. In this study we have immunoprotective functions of opsonization and immune examined the contribution of activated terminal complement complex clearance (10). proteins to the pathogenesis of the lupus-like autoimmune In this study, we have directly examined the involvement of disease. Female NZB/W F1 mice were treated with a mono- complement in the pathogenesis of the glomerulonephritis in clonal antibody (mAb) specific for the C5 component of CS sufficient NZB/W F1 mice, using a mAb specific for murine complement that blocks the cleavage of C5 and thus prevents C5 (11). We show that continuOus treatment with an anti- the generation of the potent proinflammatory factors C5a and murine C5 mAb results in marked amelioration of the course C5b-9. Continuous therapy with anti-C5 mAb for 6 months of renal disease and in dramatic prolongation of survival. resulted in significant amelioration of the course of glomer- These results demonstrate an important role for activated ulonephritis and in markedly increased survival. These find- terminal complement components in the immune complex- ings demonstrate an important role for the terminal comple- mediated inflammatory disease of NZB/W F1 mice. ment cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated CS inhibition may be a MATERIALS AND METHODS useful approach to the therapy of immune-complex glomerulonephritis in humans. Animals. Female NZB/W F1 mice (8- to 12-weeks old) were purchased from The Jackson Laboratory and were maintained under pathogen-free conditions. The NZB/W F1 mouse develops a spontaneous autoimmune Antibodies and Treatment. The anti-mouse CS hybridoma disease process with striking similarities to human systemic BB5.1 as well as the control murine anfti-human C8 hybridoma lupus erythematosus (SLE). In female NZB/W F1 mice, the 135.8 were described previously (9). Both hybridomas were production of IgG antinuclear antibodies, including antibodies grown as ascites in athymic mice and the antibodies were to double-stranded DNA (dsDNA), is associated with the purified from ascites by protein A affinity chromatography development of a severe immune complex-mediated glomer- followed by elution with ImmunoPure IgG elution buffer ulonephritis that results in death from renal failure in virtually (Pierce) and dialysis against Tris-buffered saline. Concentra- all animals by 12 months of age (1). tions of purified antibodies were.determined at O.D. 280 with Although various studies have explored the factors respon- a Beckman DU-640 spectrophotometer. At 18 weeks of age, sible for the onset of autoimmunity in these mice (2), little is mice were begun on biweekly treatments with 1 mg of either known regarding the pathogenic mechanisms of the renal anti-CS or control mAb administered intraperitoneally. Be- disease following immune complex deposition. For example, ginning at 26 weeks of age, the frequency of treatments was one hallmark of immune complex-initiated inflammation is the increased to three times per week and from 32 weeks onward activation of the complement cascade through both the clas- daily treatments were initiated. Mice were bled every 2 weeks sical and alternative pathways (3). In NZB/W F1 mice, as in for determination of serum hemolytic activity. Hemolytic human SLE, the production of autoantibodies and consequent assays were performed as described previously (9). The 100% tissue deposition of immune complexes result in local and value for complement-dependent serum hemolytic activity was systemic complement activation sufficient in magnitude to determined using normal mouse serum (Sigma). cause a marked reduction in serum complement-dependent Assays. Urine protein levels were determined three times hemolytic activity (4). This observation clearly implicates the per week by colorimetric analysis using dipsticks (Chemstrip complement system in the pathogenesis of the immune com- 2GP, Boehringer Mannheim) and quantitated according to the plex nephritis. However, studies in NZB-derived mouse strains following parameters: trace; 1+, 30 mg/dl; 2+, 100 mg/dl; 3+, deficient in the C5 component of complement have not 500 mg/dl. Standard ELISA assays to measure the serum

The publication costs of this article were defrayed in part by page charge Abbreviations: SLE, systemic lupus erythematosus; H&E, hematox- payment. This article must therefore be hereby marked "advertisement" in ylin and eosin; FcR, Fc receptor. accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 8563 Downloaded by guest on September 29, 2021 8564 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) levels, specificity, and isotype of anti-DNA antibodies were injected intradermally with 100 gg of purified rabbit anti-BSA performed as previously described (12). Briefly, 96-well flat IgG or anti-OVA IgG (Cappel) or buffer alone in 50 ,lI. bottom ELISA plates (VWR Scientific) were coated with Intradermal injections of anti-BSA IgG were also performed dsDNA (Sigma) or single-stranded DNA (Sigma) at 4°C in animals that had not received intravenous antigen as overnight and were then blocked with bovine serum albumin controls. After 4 hr, the areas of the macroscopic skin lesions (BSA) before incubation with various dilutions of serum were determined by multiplying the maximal transverse widths samples obtained from NZB/W F1 mice. The plates were (mm) in two perpendiculkr directions. Skin biopsies were washed and then incubated with horseradish peroxidase- taken through the center of the lesions, fixed in 10% buffered coupled goat anti-mouse Ig (Zymed). For isotype analysis of formalin, H&E-stained, and then examined for edema, hem- anti-dsDNA antibodies, horseradish peroxidase-coupled anti- orrhage, and inflammatory cell infiltrates. bodies to mouse IgM, IgA, IgGl, IgG2a, IgG2b, or IgG3 derived from a murine Ig isotype subtyping kit (Boehringer Mannheim) were added to the dsDNA coated wells. Following RESULTS incubation with substrate (O-phenylene-D-diamine for the Inhibition of Complement in NZB/W F1 Mice. To examine horseradish peroxidase-coupled anti-mouse Ig antibody; and the role of activated terminal complement components in the 2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate] for the progression of autoimmune disease in NZB/W F1 female mice, horseradish peroxidase-coupled antibodies in the Ig isotype 4-month-old animals were begun on biweekly treatments with determination kit), the plates were read at O.D. 490 nm and either anti-C5 or an isotype matched control mAb. We have 405 nm, respectively. Baseline levels of anti-dsDNA and previously reported that the anti-C5 mAb blocks the genera- anti-single-stranded DNA antibodies were determined using tion of both CSa and CSb-9 (9). In vivo inhibition of comple- sera from 5-week-old NZB/W F1 female mice before the onset ment by anti-C5 mAb was ascertained by serial measurement of signs of autoimmune disease. The serum titers of anti-DNA of complement-dependent serum hemolytic activity. The mAb antibodies in anti-C5 and control mAb-treated animals were treatments, continued until the mice were 40 weeks, were measured at 18, 28, and 32 weeks. The results for each group titered thereafter to maintain serum hemolytic activity at a were recorded as the fold increase in the number of O.D. units level less than 10% of that of normal control mouse serum relative to the common baseline titer measured on the serum (Fig. 1A). At the time of initiation of therapy, all mice had low from the 5-week-old NZB/W F1 mice. but measurable levels of circulating anti-dsDNA antibodies Renal Histopathology. The kidneys from euthanized control (Fig. 1B). mAb-treated, anti-C5 mAb-treated and young untreated mice Anti-C5 mAb administration was able to sustain comple- were fixed in 10% buffered formalin. The tissue was then ment inhibition in vivo for the entire 6-month period of processed and embedded in paraffin with a VIP tissue pro- treatment, as measured by reduced serum hemolytic activity cessor (Miles). Tissue sections (5 gm) were stained with (Fig. 1A). In contrast to anti-C5 mAb-treated animals, the hematoxylin/eosin (H&E) or with periodic acid Schiff using serum hemolytic activity of the control mAb-treated mice was standard methodology. normal at the outset of the study and then gradually declined Quantitative analyses of mesangial matrix deposition, glo- to less than 10% of normal levels by 40 weeks, presumably merular crescent formation, and deposition of tubular casts, secondary to systemic consumption of complement after wide- representative features of the renal histopathology in the spread tissue deposition of immune complexes (Fig. 1A). The NZB/W F1 model of lupus glomerulonephritis, were per- decline in hemolytic activity in the sera of the control animals formed on tissue from control-mAb-treated mice, anti-C5 correlated with elevated titers of anti-dsDNA antibodies mea- mAb-treated mice, and untreated 18-week-old NZB/W F1 sured over the same period of time (Fig. 1B). Both anti-C5 and mice (to determine the baseline values at the time treatment control mAb-treated mice produced comparable amounts of was begun). All samples were randomly chosen from equiva- anti-dsDNA antibodies, as measured relative to the same lently sectioned specimens processed from mice and were read baseline serum sample (Fig. 1B). A much smaller, but detect- in a blinded fashion by two independent investigators. For able, increase in the level of anti-single-stranded DNA anti- analysis of mesangial matrixvolume and crescent formation, 10 bodies was also observed (Fig. 1B). Moreover, consistent with randomly selected H&E-stained glomeruli were analyzed per previous reports (13), the predominant isotype of the anti- mouse. Images were captured using a JVC TK-1070U video dsDNA antibodies produced was IgG2a (Fig. 1C). The isotype system through a 40x objective lens and were quantitated with pattern of anti-dsDNA antibody production was unaffected by IMAGE-PRO PLUS software (Media Cybernetics, Silver Spring, anti-C5 mAb therapy (Fig. 1C). In addition, quantitative MD). To analyze mesangial matrix deposition, the perimeter immunofluorescence demonstrated equivalent levels of im- of the glomerular mesangial matrix from each hematocylin/ mune complex and C3 deposition in the glomeruli of anti-C5 eosin (H&E)-stained glomerulus was traced and the area and control mAb-treated mice (data not shown). determined by performing pixel counts. Mesangial cell areas Amelioration of Immune Complex Glomerulonephritis and were automatically excluded from the measurements using the Prolongation of Survival by Anti-C5 Therapy. The influence color differentiation parameters of the count/size command of of mAb-mediated C5 inhibition on the course of NZB/W F1 the software. Similarly coded glomerular images were also immune complex nephritis was examined both clinically and analyzed for the presence of crescent formation, and the histopathologically. A marked delay in the onset of severe percentage of glomeruli with crescents was calculated for each proteinuria, defined as equal to or greater than 500 mg/dl animal. Quantitative analysis of the number of tubular casts (.3+), was achieved in anti-C5 mAb-treated mice relative to was performed by counting all the tubules in one coded 20X control mAb-treated animals and untreated age-matched field obtained from a site on the renal cortex directly opposite NZB/W F1 mice (Fig. 2A). Whereas all of the control mAb- the renal pelvis and determining the percentage of enlarged treated and untreated mice had developed proteinuria accom- tubules filled with casts relative to the total number of tubules panied by marked total body edema by 32 weeks, no anti-C5 identified. Two independent counts by separate observers mAb-treated animals developed proteinuria until 33 weeks, were performed on each kidney and the mean percentage of and a significant percentage of these mice maintained normal tubules filled with casts was calculated. renal function without evidence of proteinuria throughout the Arthus Reactions. Reverse passive Arthus reactions were treatment period (Fig. 2A). performed on anesthetized anti-CS mAb-treated and control Coincident with ameliorating the clinical signs of severe mice. The mice were shaved and injected intravenously with 20 immune complex nephritis, CS inhibition was associated with mg/kg of bovine serum albumin (Miles). They were then a dramatic prolongation of survival. Nearly 80% of anti-CS Downloaded by guest on September 29, 2021 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8565

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10 C O. 2 Age (weeks) 18 Wks 8 Wks FIG. 2. (A) Anti-C5 mAb therapy delays onset of proteinuria in < &- A3 12 Wks NZB/W F1 mice. Serial measurements of urine protein in untreated, control mAb-treated, and anti-C5 mAb-treated animals were performed. The data are presented as percentage of animals with .3+ urine protein. (B) Prolongation of survival following anti-C5 mAb z 2 treatment of NZB/W F1 autoimmune disease. Shown are the per- o-2 centage of surviving animals in anti-C5 treated (n = 20), control = = _ . i . i ...... mAb-treated (n 27), and mAb-untreated (n 8) groups of animals .i at different ages. (A and B) Untreated, (A); control mAb-treated, (0); Ig Subypes m A mAb anti-C5 mAb-treated, (0). Control mAb Anti-CS mAb of renal tissue from anti-C5 mAb-treated mice with .3+ FIG. 1. (A) In vivo inhibition of complement by anti-C5 mAb proteinuria also demonstrated only mild abnormalities of administration. Female NZB/W F1 mice were treated with anti-C5 or tubular and glomerular architecture, with modestly increased isotype matched control mAb beginning at 18 weeks as described. Sera mesangial matrix deposition (Fig. 3D). from both treatment and control groups were tested for complement dependent hemolytic activity every 14 days until 40 weeks. (0), Control A systematic quantitation of the histologic changes in the mAb-treated; (a), anti-C5 mAb-treated. (B) Progressive age-related kidneys of control mAb vs. anti-C5 mAb-treated mice was elevation of serum anti-dsDNA and anti-single-stranded DNA anti- performed by examining multiple tissue sections from each body titers in NZB/W F1 mice. Antibody titers (O.D. units) were group of animals. As demonstrated in Fig. 4A, the amount of determined at 18, 28, and 32 weeks, and recorded as fold increase mesangial matrix deposition in C5-inhibited mice with normal relative to a common baseline titer performed on serum from 5-week- renal function (< 1+ proteinuria) was markedly reduced com- old NZB/W F1 mice as described. Solid symbols, control mAb-treated; pared with control mAb-treated mice (P < 0.001), and was open symbols, anti-C5 mAb-treated; squares, anti-single-stranded DNA; circles, anti-dsDNA. (C) Titers of various isotypes of anti- only modestly increased relative to the volume of mesangial = dsDNA antibodies measured in sera of control mAb-treated and matrix in normal glomeruli from young NZB/W F1 mice (P anti-C5 mAb-treated mice at three different time points. Assays were 0.13). Stratification of anti-C5 mAb-treated mice into groups performed as described. having low (-1+) vs. high (.3+) levels of urine protein showed less mesangial matrix expansion in the treated animals mAb-treated mice were still alive at 40 weeks, in contrast to without proteinuria (P < 0.01) compared with those with <5% of animals treated with control mAb and none of the significant levels of urine protein. Furthermore, consistent age-matched untreated mice (Fig. 2B). Histopathologic exam- with the morphology shown in Fig. 3, this analysis also revealed ination was performed on renal tissue from anti-C5-treated, that even those anti-C5 mAb-treated animals with .3+ pro- control mAb-treated, and young untreated animals (Fig. 3). teinuria had considerably less expansion of the mesangial Fig. 3A shows a representative glomerulus from an H&E matrix than control mAb-treated animals excreting equivalent stained kidney of an 18-week-old NZB/W F1 mouse before the amounts of urine protein (P < 0.001) (Fig. 4A). Quantitative onset of signs of renal disease, illustrating normal glomerular analyses of glomerular crescent formation and the incidence of and tubular architecture. Fig. 3B is representative of glomeruli tubular casts showed similar results (Fig. 4 B and C). Crescents from control mAb-treated mice with 23 + urine protein, were present in nearly 60% of glomeruli from control mAb- characterized most dramatically by marked expansion of the treated animals, in contrast to less than 10% of glomeruli from mesangial matrix with amorphous eosinophilic staining mate- anti-C5-treated animals without significant proteinuria (P < rial and a large epithelial crescent (Fig. 3B). In contrast, 0.001, Fig. 4B). Similar dramatic differences between the two examination of renal tissue from age-matched anti-C5 mAb- groups were observed with respect to the formation of tubular treated mice without detectable proteinuria revealed normal casts (P < 0.001, Fig. 4C). Again, stratification of anti-C5 appearing tubular and glomerular architecture, with only mAb-treated mice into groups having low (c1 +) vs. high minimal mesangial expansion. (Fig. 3C). Interestingly, analysis (-3 + ) levels of urine protein showed significantly less crescent Downloaded by guest on September 29, 2021 8566 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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E 80 C FIG. 3. Histopathologic examination of kidneys from anti-C5 mAb-treated and control female NZB/W F1 mice. Representative O 60 - H&E stained kidney sections are shown from the following animals. (A) Eighteen-week-old mice before the onset of autoimmune disease, 0o illustrating normal glomerular architecture. (B) Control mAb-treated 40O animals with .3+ proteinuria, showing extensive mesangial matrix deposition and crescent formation. (C) Anti-C5 mAb-treated mice ._ with <1 + proteinuria, illustrating normal glomerular architecture and 2P a minimal increase in mesangial matrix deposition. (D) Anti-C5 mAb-treated mice with .3+ proteinuria, demonstrating a modest increase in mesangial matrix deposition. (Bar = 25 t,m.) (x270.) Proeatmenua r3+ An3+ A1 + 51+ formation (P < 0.004), as well as tubular cast deposition (P < mAb treatment Control Antl-05 Anti-C5 Untreated 0.003) among anti-C5 mAb-treated animals with proteinuria 18 weeks relative to control mAb-treated mice with comparable levels of FIG. 4. Quantitation of mesangial matrix deposition, crescent urine protein (Fig. 4 B and C). Thus, taken together the data formation, and tubular casts in glomeruli from control mAb-treated, in Figs. 3 and 4 demonstrate that complement inhibition at C5 anti-C5 mAb-treated (>3+ urine protein), anti-C5 mAb-treated significantly reduces the severity of the renal histopathologic (-1+ urine protein), and mAb-untreated 18-week-old female lesion in NZB/W F1 mice, even in the presence of proteinuria. NZB/W F1 mice. Kidney sections were processed from euthanized Evaluation of Arthus Reactions in Anti-C5 mAb-Treated animals and images were captured and analyzed in blinded fashion as described. Vertical bars represent standard errors of the mean. NZB/W F1 Mice. Although the abrogation of renal disease in Statistically significant differences between the groups were confirmed the anti-C5 mAb-treated animals was associated with sus- by the two-tailed Student's t test. (A) Quantitation of mesangial matrix tained complement inhibition, it remained possible that the deposition in 10 randomly selected glomeruli from control mAb- antiinflammatory effect of the mAb administration could have treated (n = 15), anti-C5 mAb-treated (>3+ urine protein, n = 10), resulted at least in part from an alternative mechanism. For anti-C5 mAb-treated ( 1 + urine protein, n = 7), and mAb-untreated example, mAb interaction with circulating C5 could have 18-week-old female NZB/W F1 mice (n = 6). Matrix volumes were enhanced the of the Fc of the mAb to Fc determined as described and are represented as mean pixel counts. (B) binding portion Quantitation of the percentage of glomeruli with crescent formation. receptors (FcR) on inflammatory leukocytes, resulting in Ten randomly selected glomeruli were examined per mouse. (C) systemic FcR blockade. In this light, Ravetch and colleagues Quantitation of the percentage of renal tubules containing protein (14, 15) have recently demonstrated that the direct activation casts. The data are based on the number of tubules with casts as a ofFcR-bearing cells represents a primary mechanism initiating percentage of the total number of tubules identified per low power several antibody-mediated inflammatory responses, including (20x) field (between 30 and 75 tubules per animal as described). The the immune complex-triggered inflammation of the murine determinations of crescent formation and tubule deposition were Arthus reaction. performed in blinded fashion as described on control mAb-treated To address this reverse cutaneous Arthus (n = 8), anti-C5 mAb-treated (-3+ urine protein, n = 7), anti-C5 possibility, passive mAb-treated (-<1+ urine protein, n = 7), and mAb-untreated 18- reactions were performed on anti-C5 as well as control mAb- week-old female NZB/W F1 mice (n = 6). treated NZB/W F1 mice. If the anti-C5 mAb administration had induced systemic FcR blockade, a marked attenuation of complement-dependent serum hemolytic activity (Fig. 5A), the Arthus reaction should be observed. NZB/W F1 mice were they manifested Arthus reactions similar in magnitude to those therefore treated for two weeks with daily 1-mg doses of either ofthe control animals (Fig. SB). The specificity of the reactions the anti-C5 or control mAb, at which time the Arthus reactions was ascertained by the absence of measurable inflammatory were induced. The animals were injected intravenously with 20 responses following intradermal injection with anti-OVA IgG mg/kg of BSA followed by intradermal injections of 100 ,ug of or with buffer alone (Fig. 5B), or following intradermal anti-BSA IgG, anti-OVA IgG, or buffer alone, and the resulting injection of the anti-BSA IgG to mice that had not received cutaneous inflammatory responses were evaluated after 4 hr. intravenous BSA (data not shown). Histologically, the Arthus The results are shown in Fig. 5 and demonstrate that while reactions in both the anti-C5 mAb-treated and control animals the anti-C5 mAb-treated mice had profound inhibition of were also comparable, characterized by edema and intense Downloaded by guest on September 29, 2021 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8567 Control mAb Anti-C5 mAb inflammatory cell infiltrates comprised predominantly of neu- trophils (Fig. 5C, panels 2 and 4). No significant inflammatory z 1X0 A response was observed in either group of animals following - intradermal injection of the specificity control anti-OVA IgG .> 80 (Fig. SC, panels 1 and 3). The marked Arthus reactions observed in the C5 inhibited mice relative to the control animals clearly indicated that anti-C5 mAb administration had not resulted in functionally significant FcR blockade.

I20 DISCUSSION The progression of fatal glomerulonephritis in C5 sufficient 0 NZB/W F1 mice is associated with rising titers of autoanti- E 120 Tr bodies, deposition of immune complexes, and marked local B and systemic complement activation. Here we have shown that inhibition of the complement cascade with a C5-specific mAb 0N44A markedly ameliorates the course of nephritis and prolongs X 80 survival, clearly implicating the products of terminal complement activation in the inflammatory process leading to renal E o. failure and death. 80 Cleavage of C5 releases CSa, a potent anaphylatoxin and 20 chemotactic factor, and leads to the formation of the lytic terminal complement complex, CSb-9. Both CSa and CSb-9 also have pleiotropic cell activating properties, as they have antEBSA: been shown to amplify the release of downstream inflamma- anti-OVA: + - tory factors such as hydrolytic enzymes, reactive oxygen spe- buffer: + -- cies, arachidonic acid metabolites, as well as various cytokines (16-19). While preventing the generation of these proinflam- 3 s matory terminal complement components, mAb-mediated c inhibition of the complement cascade at C5 preserves the ability to generate C3b, which is critical for opsonization of many pathogenic microorganisms as well as for immune complex solubilization and clearance (10). Retaining the ca- pacity to generate C3b would appear to be a particularly important aspect of a therapeutic approach to complement inhibition in an inflammatory disease like SLE, where both increased susceptibility to infection and impaired clearance of immune complexes are preexisting clinical features of the disease process (20). Our results suggest that complement activation following tissue deposition of autoantibody-containing immune complexes plays a prominent role in initiating the renal inflammatory response in NZB/W F1 mice. Several other immuno- therapeutic approaches to the treatment of NZB/W F1 autoimmune disease, such as blockade of T cell costimulation through the B7 pathway (21), or antagonism of the activity of cytokines such as IL-10 (22), have been associated with suppression of autoantibody production, and thus also support a role for humoral immunity in disease pathogenesis. The recent studies of Ravetch and colleague (14) with FcR-deficient mice derived by germline mutation of the FcR- FIG. 5. Evaluation of reverse passive Arthus reactions in anti-C5 associated y chain indicated that the murine Arthus reaction mAb-treated and control mice. Young (6-week-old) NZB/W F1 mice is driven primarily through direct activation of FcR-bearing were treated for two weeks with daily i.p. 1 mg doses of anti-C5 or cells, with complement serving a role in the amplification of control mAb. Arthus reactions were then performed as described. (A) the ensuing inflammatory response. Consistent with these Serum hemolytic assays confirmed that anti-C5 treatment had resulted findings, we have found that significant Arthus reactions can in profound systemic complement inhibition. (B) The size of each be induced in anti-C5 mAb-treated NZB/W F1 mice, and Arthus reaction was determined by multiplying the two widest per- a role pendicular transverse diameters (mm) of the lesion. Mice were in- conclude that terminal complement may play secondary jected with 20 mg/kg BSA intravenously, followed by intradermal in this murine model of inflammation. However, the data in this injection with 100 ,ug of rabbit anti-BSA IgG or rabbit anti-OVA IgG, report, as well as our previous demonstration that C5 inhibition or with buffer alone. The lesions were evaluated at four hr. A total of resulted in marked amelioration of the course of collagen- three lesions were evaluated per animal, with nine animals per group. induced arthritis (9), further indicate that activated terminal No inflammatory responses were observed following intradermal injec- complement components are primary mediators of inflammation tion with anti-OVA IgG or with buffer alone. (C) Histological evaluation following immune complex deposition in the kidney or joint. of Arthus reactions. All the mice were injected with BSA 20 mg/kg Thus, the predominant pathway through which an immune intravenously. Representative H&E stained sections are shown from an inflammatory response may depend in part control mice injected intradermally with anti-OVA IgG (panel 1) or complex triggers anti-BSA IgG (panel 2), and anti-C5 mAb-treated mice injected with on the tissue site of immune complex deposition. anti-OVA (panel 3) or anti-BSA (panel 4). There is similar intensity of the Previous reports have shown that several C5 deficient inflammatory responses elicited by anti-BSA injection in both groups, NZB-derived inbred mouse strains (5, 6), as well as C5 noted both intradermally and subcutaneously. (X47.) deficient (NZB x SWR) F1 mice (7), develop immune complex Downloaded by guest on September 29, 2021 8568 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) nephritis, and have been cited as evidence against a role for 3. Colten, H. R. (1994) Nature (London) 371, 474-475. terminal complement in NZB/W F1 autoimmune disease. In 4. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., this regard, we would suggest that analyses ofmice congenitally Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B. & deficient in C5 may not invariably reflect the actual function Dixon, F. J. (1978) J. Exp. Med. 148, 1198-1215. 5. Lanier, B. G., McDuffie, F. C. & Holley, K. E. (1971)J. Immunol. of terminal complement in inflammatory responses of animals 106, 740-746. with an intact complement cascade. This notion is based in part 6. Rudofsky, U. H., Evans, B, D., Balaban, S. L., Mottironi, V. D. & upon studies in gene-targeted mice, which have shown that Gabrielsen, A. E. (1993) Lab. Invest. 68, 419-426. mutant animals can compensate for the loss of a critical gene 7. Datta, S. K., Patel, H. & Berry, D. (1987) J. Exp. Med. 165, product to produce a normal phenotype (23). For example, this 1252-1268. could occur when there are distinct molecules that can mediate 8. Rinder, C. S., Rinder, H. M., Smith, B. R., Fitch, J. C. K., Smith, functions similar to those of the targeted gene product (24, 25). M. J., Tracey, J. B., Matis, L. A., Squinto, S. P. & Rollins, S. A. In this light, given the critical role of the complement system (1995) J. Clin. Invest. 96, 1564-1572. in host defense and it is 9. Wang, Y., Rollins, S. A., Madri, J. A. & Matis, L. A. (1995) Proc. immunoregulation, possible that C5 Natl. Acad. Sci. USA 92, 8955-8959. deficient mice compensate for the inability to generate CSa 10. Liszewski, K. M. & Atkinson, J. P. (1993) in Fundamental Im- and CSb-9 by up-regulating the activity of functionally redun- munology, ed. Paul, W. E. (Raven, New York), pp. 917-939. dant alternative inflammatory mediators. These in turn could 11. Frei, Y., Lambris, J. D. & Stockinger, B. (1987) Mol. Cell. Probes play a role in terminal complement-independent inflamma- 1, 141-149. tory responses. Mechanisms of immune complex-triggered 12. Jabs, J. A., Burek, L. C., Hu, Q., Kuppers, R. C., Lee, B. & inflammation that could promote the development of renal Prendergast, R. A. (1992) Cell. Immunol. 141, 496-507. disease in the absence of terminal complement include en- 13. Behar, S. M., Corbet, S., Diamond, B. & Scharff, M. D. (1989) hanced activity of the normally weak anaphylatoxin C3a, or the Int. Rev. Immunol. 5, 23-42. 14. Sylvestre, D. L. & Ravetch, J. V. (1994) Science 265, 1095-1098. direct activation of FcR-bearing cells discussed above. It is 15. Clynes, R. & Ravetch, J. V. (1995) Immunity 3, 21-26. possible that these mechanisms could also account for the 16. Daniels, R. H., Houston, W. A. J., Petersen, M. M., Williams, renal disease observed in a percentage of the anti-C5 mAb- J. D., Williams, B. D. & Morgan, B. P. (1990) Immunology 69, treated mice (Fig. 2). However, even in the anti-C5-treated 237-242. mice that developed proteinuria, the renal histopathology was 17. Gerard, C. & Gerard, N. P. (1994) Annu. Rev. 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