complement. the terminal membrane attack complex of Activation of ...

0022-1767/87/1388-2473802.00/0

Voi. 138.2473-2480. No. 8.Aprll15.1987 Prlnted In U.S.A.

THEJOURNAL OF h M U N O t o C K Copyright0 1987 by The Amerlcan AssOclatlon of lmmunolcglsts

ACTIVATION OF GLOMERULARMESANGIALCELLS BY THETERMINAL MEMBRANE ATTACK COMPLEX OF COMPLEMENT' DAVID H.LOVETT,2"GERTRUD-MARIAHAENSCH,' M. GOPPELT,'KLAUSRESCH," DIETHARD GEMSA'

AND

From *The Institut fur Molekularpharmakologie, Medizinische Hochschule Hannover. Hannover, FRG; the +MedicalService. Seattle VAMC,Seattle. WA; the 'Institutefur Immunologie, Uniuersitat Heidelberg, Heidelberg, FRG: and the 'Institutfur Immunologie, Universitat Marburg, Marburg.FRC

(MAC) of C5b-9 was closely linked to the development of Treatment of cultured renal glomerular mesangial cells (MC) with nonlytic concentrationsof the puri- glomerular injury in experimental membranous nephroof the terminal membrane pathy and in the heterologous phase of antibasement fied components (C5b-9) attack complex (MAC) of complement induced sig- membranenephritis (5, 6). Subsequently, glomerular nificant functional alterations characteristic of cel- deposition of the MAC in several experimentalmodels of lularactivation.C5b-9-treated MC releasedlarge GN was shown to correlate withdevelopment the of injury quantities of primarily vasodilatory prostaglandins.(7, 8).MAC deposits were also found to be present in the In addition, the secretion of an MC-derivedauto- mesangium inmodels of glomerulosclerosis not mediated growthfactor (MC interleukin 1) wasgreatlyenby immune mechanisms (9). Glomerular deposition of hanced. Examinationof the action of C5b-9 on MC MAC, particularlyin the mesangium. has been docuphospholipidmetabolismindicatedthatcomplemented in several human disorders, including systemic mentinduced theactivation of phospholipases, lupus erythematosus, IgA nephropathy, and membranoleading to quantitative changes in the fatty acid profile of MC membrane phospholipids. These find- proliferative GN (10,1 1). Significant mesangial MAC depings demonstrate that cultured MC are highly re- osition was also observed in biopsies from patients with sponsive to nonlytic concentrations of the C5b-9 diabetes mellitus and hypertension, especially in areas of mesangial sclerosis(1 1). complex, andsuggest that the mesangial deposition Although indicating an association ofMAC deposition of the MAC in many forms of glomerular disease, with resultant cellular activation, may play major a with glomerular disease, these observations do not prorole in the hemodynamic and cellular proliferative vide direct evidence for a causative role of the MAC in the induction of glomerular injury. Given the constraints events characteristicof these disorders.

Complement deposition is a histologic hallmark of immune-mediatedglomerulonephritis (GN).3 It had been previously thought that theinflammatory action of complement was indirect.After activation of complement and release of chemoattractants, glomerular injury was attributed to the release of lysosomal hydrolases by infiltrating polymorphonuclear leukocytes (1-3).More recent evidence, obtained primarily from experimental models of GN, has demonstrated a direct, or cell-independent, role of complement in theinduction of glomerular injury (4).These findingswere further extended by observations suggesting that the terminal membrane attack complex Received for publication August 27, 1986. Accepted for publication December 19. 1986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertlsernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by grants from the Deutscheforschungsgemeinschaft (Ge 35415-2.Re 28119-1).research funds of the Veterans Administration, and by a fellowship to D. H. L. from the Alexander von Humboldt Stiftung. Bonn. FRG. *To whom all correspondence shouldbe addressed at: Medical Service 1 1 lA,Seattle VAMC, 1660 S . Columbian Way, Seattle. WA. 98108. Abbreviations used in this paper: MC, mesangial cells; MAC, membrane attack complex: GN.glomerulonephritis: MC-TAF. MC-thymocyteactivating factor: DMEM. Dulbecco's modified Eagle's medium: IF, immunofluorescence: PG. prostaglandin; AA. arachidonic acid: TLC. thinlayer chromatography: PL. phospholipids; PC, phosphatidyl choline: PI. phosphatidyl inositol: PE, phosphatldyl ethanolamine; PS, phosphatidyl serine.

of animal models of GN, we elected to determine the effects of the purified terminal complement components C5b-9 on the biologic properties of defined populations of cultured glomerular mesangial cells (MC). MC are the source of a number of inflammatory mediators. These includeprostaglandins (PG) and a polypeptide closely resembling macrophage interleukin1 (IL 1)(12). The MCderived IL 1 was recently purified to homogeneity and was foundto act both as a stimulant of MC PG synthesis and as an autogrowth factor (13).4 The findings reported here demonstrate that nonlytic concentrations of C5b-9 exert a potent stimulatory action onPG and IL 1 release by MC, events associated with significant alterations in cellular phospholipid metabolism. Theseobservations provide further support for the hypothesis that themesangial deposition of terminal complement C5b-9 plays an active role in the development of glomerular injury. MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats, 125 to 150 g (Versuchstieranstalt. Hannover, FRG). were used to provide thymocytes andMC. Media. Actively growing MC were maintained in Dulbeccosmodified Eagles medium (DMEM) supplemented with 20% heat-inactivated fetal calf serum (FCS: GIBCO. Paisley, Scotland), 100 U/ml penicillin. 100 &ml streptomycin, and300 &/ml glutamine (growth medium). Preparation of cells. Primary cultures ofMC were obtained as outgrowths from collagenase-treated glomerular remnants derived

€I..

'Lovett, D. K.Resch. and D. Gemsa. 1986.Interleukin I and the glomerular mesangium. 11. Monokine stimulation of mesangial cell prostanoid secretion. Subrnlttedfor publlcatlon.

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COMPLEMENT

ACTIVATION

from bloodfree. uerfused kidnevs as reported in detail previously (12, 14). Exponential growth was maintained in growth medium in a humidified 5% COZ atmosphere. MC appeared as homogeneous outgrowths of stellate cells growing in interwoven bundles. Immunofluorescence (IF) staining &ealez prominent intracellular myosin fibrils. as well as significant amounts of types IV and V collagen and fibronectin in the extracellular matrix (14). IF staining was negative for Ia surface antigen. common leukocyte antigen, and Factor VIII. Cells with the morphologic or functional features of macrooha@es were not present. MC were passaged at 1:6 split ratios with &yp&/ EDTA and were used between the fourth and sixth passages for complement stimulation studies. Preparation of complement. C5b6 was purified from normal human serum according to the method described by Yamamoto and Gewurz (15). Human C7. C8, and C9 were isolated as reported by Hammer et al. (16). The functional activity of the components was tested in a modified reactive lysis system with chicken erythrocytes used as target cells (17). Activitv was calculated as CHso units fcomplement hemolytic titer 50%). i U being the amount I$ component with which 50% of the erythrocytes could be lvsed. Before use, the purified components were absorbed with polymyxin B-Sepharose (100 pl packed beads/ml component), and were free of detectable endotoxin contamination (CO. 1 rig/ml) when assessed with the amebocyte lysate assay (E-toxate , Sigma Chemical Co.. St. Louis. MO):. IL 1 assay. Qu&tation of &e amounts of MC-derived IL 1 (or MC-thymocyte-activating factor (MC-TAF)) released in response to complement components was pe;formed as reported previously, in which the proliferative responses of lectin-primed rat thymocytes were used (12). IL 1 activity is expressed in units derived from probit analysis, which compared the half-maximal proliferative responses of test preparations to a rat peritoneal macrophage standard assigned an arbitrary activity of 100 U/ml. A 50% proliferative response was generally achieved with 5 to 10 U/ml of IL 1. Control experiments demonstrated that the purified complement components in the concentrations used in these studies had no effect on the thymocyte proliferative response to known quantities of purified IL 1. Biochemical characterization of mesangial cell IL 1 activitv released in response to complement was unciertaken by using an approach outlined in detail previously (13). Cultures of MC were treated with nonlytic concentrations of C5b-9 as detailed below. After 24 hr of incubation. the conditioned medium was harvested and was centrifuged at 400 X G for 10 min. The conditioned medium was subsequently ultra/diafiltered against 100 mM NaCl, 50 mM Tris/HCl. pH 7.4, 0.01% polyethylene glycol (m.w. 4000 to 6000). and was concentrated to l/50 of the original volume in an Amicon chamber using a YM-10 membrane (Amicon. Danvers, MA). This material was then applied to a calibrated column of Sephacryl S-200 SF (Pharmacia, Freiburg, FRG) equilibrated with the same buffer. Fractions eluting between 12,500 and 16,000 m.w. were pooled, were ultra/diafiltered against 25 mM imidazole HCl, pH 7.4, and were applied to a chromatofocusing column (Pharmacia). The column was developed with a linear gradient of a l/8 dilution of Polybuffer exchanger. pH 4 (Pharmacia). Fractions’of 7 ml were collected and the pH was determined immediately. Thereafter. the fractions were dialyzed against phosphate-buffered saline (PBS), were sterilized by filtration, and the amount of IL 1 activity was determined in the thymocyte assay. PC assays. Radioimmunoassays (RIA) were used to determine the amount of PGE, thromboxane Ba. and 6-keto-PGF-1-a present in the supernatants of stimulated MC, using antibodies and methods described in detail previously (18, 19). There was no detectable crossreactivity of the various antisera with the purified complement components. PG levels are expressed in pmol PG/mg cellular protein. Complement stimulation of IL 1 and PG release. MC were harvested with trypsin/EDTA and were plated at a density of 2 x lo4 cells/cm* in 16-mm plastic wells in growth medium and were allowed to grow to near confluency. At the time of study. the cell layers were washed three times with warm. serum-free medium. The terminal complement components were applied at 37°C in the following concentrations: C5b6. 10.000 IJ for 10 min: C7. 2000 U for 10 min: C8 and C9. each 2000. U for a final 10 min. DMEM supplemented with 0.5% FCS was thereafter added to make a final volume of 1.5 ml/ well. The comolement-treated cells were incubated for varvinc time periods, after which the supernatants were harvested. we;e &mtrifuged at 1000 X G for 10 min. and were stored at -20°C until assaved fo;PG and IL 1 content. Controls were incubated with medium alone. or with equivalent concentrations of complement components which had been preincubated for 1 hr at 37°C before being added to the cultures. After complement treatment, the cell lavers were washed twice with PBS, foliowed by two washes with ice-cold 5% trichloroacetic acid (TCA) to precipitate cellular proteins. The amount of protein present in each well was determined by the method of Bradford (20). using bovine serum albumin (BSA) as a standard.

OF

MESANGIAL

CELLS

Phosphollpfd (PLJ expertments. To determine the effects of terminal complement components on MC arachidonate metabolism. the cultures were prelabeled for 3 hr in serum-free medium containing 0.5% defatted BSA (Sigma) and 0.2 pCi/ml W-arachidonate (specific activitv 58 mCi/mmol: Amersham-Buchler. Braunschweig, FRG). The labeled cells were washed three times with warmed P& containing 1% defatted BSA lo remove non-incorporated label, and were then exnosed to the terminal comolement components as detailed above.At given time intervals, the cell layeri were washed twice with cold PBS, followed bv two washes with ice-cold 5% TCA. The precipitated material was scraped from the culture wells and was recovered by centrifugation at 1000 X G for 10 min. The cellular precipitate was then extracted in chloroform/methanol (2:l). The extracts were washed three times with 0.1 M KCl, followed by phase separation. The lower chloroform/methanol phase was recovered

and evaporated under a stream of nitrogen prior to PL separation by thin-layer chromatography (TLC) (see below). In other experiments. the cells were treated with terminal complement components. followed by the immediate addition of 0.2 &i/ml of “C-arachldonate. At specified time intervals, the cellular PL were recovered as described above. In both groups of experiments, aliquots of the TCAprecipitated cellular tents. Because the

proteins specific

were activity

taken and

to determine protein concounting efficiency of the

radiolabeled arachidonate were known, the results are expressed as nanomoles arachidonate/mg cellular protein. Thtn-layer chromatography {TLC). Separation of individual MC PL species was performed as detailed by Goppelt and Resch (21). In brief, the evaporated PL were resusoended in chloroform/methanol (2: 1) and were applied with an autokatic sample applicator (Camag. Muttenz. Switzerland) to plastic-backed silica gel G plates (Merck, Darmstadt, FRG) which Gad been washed previously in methanol. Dried plates were developed at 4’C in a solvent system containing chloroform/methanol/acetic acid/0.9% NaCl (50/25/8/2.5), which provided satisfactory resolution of MC phospholipids in one dimension. Representative Rf were: lysophosphatidyl choline, 0.09; sphingomyelin, 0.16; phosphatidyl choline (PC], 0.29; phosphatidyl inositol IPII. 0.39: ohosuhatidvl serine (PSI. ohosphatidyl ethanol., 0.51: ..~ amine (PE), 0.7-4. Siparateh PL were stained with iodine vapor, were identified bv comparison with Durified standards. and the incorporated radio&tivity of cut stripswas determined by beta scintillation

counting. Statistics. The data given are representative of experiments performed two or more times. The PG and IL 1 levels were determined in triplicate for each well: from 4 to 6 wells were used for each point. For the PL experiments, 4 to 6 wells were used for each point. All

data are given as mean f SEM. Where appropriate, statistical

significance

were

determined

by Students

degrees of

t-test.

RESULTS

The objective of these experiments was an assessment of the effectiveness of nonlytic concentrations of purified C5b-9 to act as a stimulant of cultured glomerular MC. The concentrations of C5b-9 used were based on prior observations with macrophages and platelets (17, 22). As previously reported (17), the concentration of C5b6 represents the limiting factor in the assembly of membraneassociated MAC in these cellular systems, and at a concentration of 10,000 U C5b6/5 X lo4 cells there was no evidence for a direct cytolytic action. Trypan blue exclusion rates at 24 hr after C5b-9 treatment were 92% + 5 vs controls with 94% f 6 exclusion (n = 12, p > 0.05). IL 1 (MC-TAF) secretion. Purified C5b-9 proved to be a potent stimulus of IL 1 release from MC [Fig. 1). Although C5b6 or C789 alone were not effective, the complete terminal complex induced a very large increase in the IL 1 secretion when assessed at 24 hr after stimulation. Although less active, C5b-8 also enhanced IL 1 release. Significantly, the addition of C5b-9 which had been preincubated for 1 hr before being added to the cultures failed to elicit an IL 1 secretory response. This corresponds to the fact that C5b-9 formed in the fluid phase loses its membrane-binding capacity (15, 17). When the time kinetics of MC IL 1 (MC-TAF) release in response to C5b-9 were examined (Fig. 2). it was found that the bulk of the IL 1 was released from 3 to 6 hr after complement

COMPLEMENTACTIVATION

2475

OF MESANGIALCELLS

Stlmulotlon of MC-TAF Release by TermlnalComplement

8

Cofnpononts

7

L

I ,

2

8

20

14

32

26

38

Fractlon number

Figure 3. Analytic chromatofocusing of complement-stimulated MC 1L 1 activity. Conditioned medium from C5b-9-stimulated MC was size-

h Control

C5b6

C 789 C5b-8 C5b-9 Prclnc C5 b-9

Figure 1 . Stimulation of MC-TAF (MC IL 1) release by terminal complement components. MC were incubated with C5b6 (10,000 U. C7. C8. and C9 (2,000 U).in the indicated combinations for 24 hr at 37OC. The amounts of MC 1L 1 present incellfree supernatants were quantitated in the thymocyte assay and are expressed as units per 0.1 mg cellular protein. Data represent mean % SEM. 'Preinc. C5b-9" denotes the effects of terminal complement components which were preincubated for 1 hr a t 3 7 Tbefore being added to the mesangial cultures.

fractionated bygel chromatography on Sephacryl 5-200 SF, and the 12,500 to 16.000 m.w. fraction was recovered. This was analyzed by chromatofocusing as detailed in Materials and Methods.The pH of each fraction was determined immediately. After dialysis against PBS. the fractions were diluted 1/10 and the amount of IL 1 activity present was determined in the thymocyte assay. The data aregiven as means [n = 4) of A cpm [3H]thymidineuptake of test materials as compared with controls (mean: 2260cpm, n = 4). S t l m u l a t l o n of PGE Release by TermlnalComplementComponents

( 6 h )

T

Tame K l n e t l c s 0 1 MC - T A F Release by C 5 b - 9

0 Conlrol

C 5b-9

Control

C5b6

C 789

C5b-9

Prelnc C 5b-9

Figure4. Stimulation of PGE release by terminal complement components. MC were incubated with varying combinations of terminal complement components as described for Figure 1. After 6 hr, the cellfree supernatants were assayed by RIA for PGE content. The results are expressed as picomoles PGE per 0.1 mg cellular protein [mean f SEM). Imel h1

activity is IL 1. PC studies. The prostanoid secretory responses of MC to C5b-9 were determined by RIA for PGE and the stable * metabolites of prostacyclin (6-keto-PGF-1-a) and thromboxane A2 (thromboxane &). Incubation of MC with C5bstimulation. Subsequent experiments which included cy- 9 for 6 hr resulted in the release of nearly 1000 pmol cloheximide in theincubation medium demonstrated that PGE/mg cellular protein (Fig. 4). Controls utilizing C5b6, this secretory phase was dependenton new IL 1 protein C789, and preincubated C5b-9 had no stimulatory action. synthesis (not shown). Similarly, at 6 hr after exposure to C5b-9, there were The biochemical properties of the IL 1 activity present significant stimulations of prostacyclin (-200 pmol/mg) in the supernatantsof C5b-9-treated MC were evaluated. and thromboxane (-20 pmol/mg) release (Fig. 5A and B). Material with a n m.w. of 12,500 to 16,000 (the m.w. of As with PGE, C5b6, C789, and preincubated C5b-9 had MC IL 1) (12, 13) was further analyzed by chromatofo- nosignificantstimulatoryaction on prostacyclin or cusing (Fig. 3).IL 1 activity was obtained predominantly thromboxane secretion when assessed at 6 hr. The efat a PI of 7.2, with a smalleramount of activity observed fects of C5b-8 on prostanoid secretion were also examat PI 4.5. These PI closely resemble those previously ined (Fig. 6). The PGE secretory response to C5b-8 was observed for rat MC IL 1 purified from nonstimulated cell approximately one-third of that obtained with the compopulations (13),and confirm that theC5b-9-stimulated plete complex (-300 pmol/mg). C5b-8 was not found to Figure2. Time kinetics ofMC-TAF[MCIL 1) release by terminal complement components. MC were incubated with terminalcomplement components as described for Figure 1 for varying time periods. Control cultures were incubated with medium alone. The results are expressed as units of interleukin activity per 0.1 mg cellular protein (mean SEM).

-

2476

COMPLEMENT ACTIVATION OF MESANGIAL CELLS

.f c

A Release by C 5 b - 9

Pa

Stlmulotlon of 6-keto-PGFlK T

Releose

b y Terminal Complement

Components

/

J

Control

W

C5b6

C 7C8 59 b - 9

/

RRIK C5b-9

Stimulation of

-

TXB2

Releoso by Terminal Complement Components

i zol -a

(6h)

1 10 5 i Release by C 5 b - 9

21.90

2

5-

m" X

I-

Control C 5 b6

C 789 C5b-9 C5b-9

FLgure 5. Stimulation of prostacyclin (A]and thromboxane (E)release by terminal complement components. Conditions for these experiments are as described for Figure 4. Silmulatlon of PG Releoso by C 5Kbh- 8l

T 3

I8

6

2L

Tlme [ h 1

Flgure 7. Time kinetics of PGE ( A ) and 6-keto-PGF-1-a (E) release in response to terminal complement components. MC were incubated with C5b-9. using concentrations as described for Figure 1 or with medium alone. The supernatantswere assayed at varying time pointsfor prostanoid content. In A, the subscript0 denotes the portion of the complementstimulated curve for whlch average rates of PGE synthesis were calculated [see Results).

Control

C 5b-8

Figure 6. Stimulation of prostanoid release by C5b-8. MC were incubated with 10,000 U of C5b6 and 2,000 U each of C7 and C8 for 6 hr. Levels of prostanoids in the supernatantswere assessed by RIA.

be an effective stimulant of prostacyclin or thromboxane release under these conditions. The time kinetics of the PG secretory response toC5b9 were evaluated in experiments in which the MC supernatants were serially harvested after stimulation with complement. As shown in Figure 7 A and B, the kinetic pattern obtained with prostacyclin (and thromboxane,

not shown) was quite different from that obtained with PGE. Prostacyclin release occurred only within 1 hr after complement stimulation. Although increased PGE secretion was detectable at 1 hr, the bulk of the PGE release occurred in a continuous fashion between 6 and 24 hr after complement exposure. The PGE secretion rate between 6 and 24 hr after complement stimulation was approximately seven times that of control cultures (91 pmol/mg/hr vs controls 12 pmol/mg/hr). The magnitude of the PG secretory response to C5b-9 was found to be dependent on the concentration of C5b6 used to treat thecells (Table I). As few a s 5000 U of C5b6 per well were sufficient to induce significant increases in PGE secretion. Higher concentrations appeared necessary to significantly enhance prostacyclin release. Although effective in stimulating PG secretion to a greater extent, a concentration of 20,000 U/well of C5b6 was associated with a large amount of cellular lysis (-30% trypan blue-positive at 6 hr).

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COMPLEMENT ACTIVATION OF MESANGIAL CELLS and A PC

TABLE I C566 Concentratfon dependence of PG secretiono Group

Control 5.000 U C5b6 10.000U C5b6 506 20,000 u c5b6

PGE pmol/mg

P G I pmol/mg

50 f 12 229 f 33b 396 f 66b f 52b

56 f 14 70 f 6.6 98 f 20b 230 f 24b

PE

Control PC C5b-9 PC 0 Control P E CSb-9 PE 0 0

"C5b6 concentration dependence of prostanold secretion. MC were incubated with varying denotedconcentrations of C5b6 along with 2000 U of C7. C8. and C9 in order to build a complete complex. After 6 hr at 37°C. the prostanold content of cellfree supernatants was determined by RIA. Data are glven as mean f SEM. p < 0.05.

PL metabolism. The effectsof C5b-9 on MC PL metabolism were examined intwo series of experiments. In the first, theMC PL were pre-labeled with '*C-arachidonate. were freed of remaining free fatty acid, and were then exposed to C5b-9. The aimof these studies wasto determine which of the individual MC PL pools were most active as sources of substrate arachidonatefor the complement-mediated PG synthesis. Representative results of these experiments are shown in Figure 8. C5b-9 induced a rapid loss in the arachidonicacid (AA) content of PC, PE, and PI whereas no significant effects on PS were found. The bulkof this decrease in AA content occurred within 15 min of exposure to C5b-9, and the concentration of labeled arachidonate in PL, PC, and PE reached a steady stateby 1 hr. ThePC pool was thesource of 83.7% f 6 of the released AA when assessed at 15min; PE and PI accounted for 12.1% f 1.4 and 4.2% f 1.2 of total released AA, respectively. Interestingly, there was a net positive flux of labeled AA into the PI pool at 2 hr after C5b-9 treatment. The labeled AA in PI presumably derived from the large amounts of arachidonate released from the PC and PEpools as a result of complement action. In a second set of experiments, the cells were stimulated with C5b-8 C5b-9 or in the presence of labeled arachidonate. Increases in PL radioactivity under these conditions thereby represent the new incorporation of AA into cellular PL. The effects of terminal complement components onthis process are presented in Figure 9 for total PL and in Figure 10A. B and C for PC, PI, and PE, respectively. Complement treatment resulted in a rapid stimulation of AA incorporation into total cellular phospholipids which was linearfor 1 hr, afterwhich timethe incorporation rates reverted to those of the controls. In the initial phase (Le., within 1 hr), C5b-8 and C5b-9 were found tobe equally effective as stimulants of arachidonate incorporation. As shownin Figure 10, there was significant incorporation of AA into PC, PI, and PE; in contrast, only 2.4 to 2.6% of the AA was incorporated into PS. As with the pre-labeling experiments, the PC pool was quantitatively the largest, and accounted for 58.6%f 4 of the incorporated AA when assessed at 2 hr after stimulation with C5b-9. Although representing only 12%of the total MCPL content (D. H. Lovett, M. Goppelt, and K. Resch, manuscript in preparation), the PIpool incorporated up to 22.8 f % of the total arachidonate, indicating a preferential flow of the fatty acid into this relatively small PL pool. DISCUSSION

Purified terminal complement components were found to influence a number ofMC biologic properties. Inasmuch that cultured MC between the fourth and sixth passages were used for these experiments, the precise

2I Effectsof C5b-9 on A A Levels In MC-Phosphol~plds 8: PI A Control P I A C5b-9 P I 1

/

O"1

B

1,

, 15

I

60

120

Tlme (m~nutasl

Figure 8. Effects of terminal complementcomponents on AA content of MC PL. MC wereprelabeled with "C-arachldonate as described in Materlals and Methods.Washed cells were then exposed to C5b-9 or to medium controls. At varying time polnts, the cellular PL were extracted and were separated by TLC. The content of radiolabeled AA remaining after complement treatment Is expressed as nanomoles per mg cellular protein (mean f SEM). Effects of C5b-9 on AA content of PC and PE (A) and PI (B).

biologic responses of these cells to terminal complement components may not be directly applicable to potential responses of MC in vivo. Careful consideration wasgiven in these experiments to the use of nonlytic concentrations of C5b-9. It is now well recognized that nucleated cells are more resistant to complement-mediated lysis than erythrocytes (23),which may be related to the very short half-life of the MAC in nucleated cell membranes (24-26). Nucleated cells can also initiate plasma membrane repair mechanisms, primarily through the stimulation of lipid synthesis (27, 28). These events may be associated withthe expression of other phenotypic properties consistent with a state of cellular activation. Our findings with complement-treated MC support the hypothesis that theterminal complement components C5b-

2478

COMPLEMENT Effects

of CSb-8 and C5b-9 of AA Into Total

lncorporatlon 20 -

0 0 .

0

ACTIVATION on PL

controt C5b-8 C 5b-9

I 60 Time (mlnuteS 1

15

I 120

Figure 9. Effects of C5b-8 and C5b-9 on incorporation of W-arachidonate into total mesangial PL. MC were incubated with either C5b-8 or C5b-9 in the presence of 0.2 &i/ml %-arachidonate for varying time periods. After extraction and TLC separation, the increases in AA content of PL were determined (mean -C SEM).

9 can act as cellular activators, and extend the recent report that complement could induce the mesangial secretion of free radical oxygen and hydrogen peroxide (29). The ability of pre-formed immune complexes to stimulate the release of macrophage IL 1 has been found to be complement-dependent (30). In the present study, it was determined that C5b-9 alone was sufficient to induce the release of mesangial IL 1. In addition to biologic activity as an endogenous pyrogen (31). we recently observed that purified MC IL 1 acts as an autocrine growth factor, thereby enhancing MC growth rates (13). The magnitude of the MC IL 1 secretory response is the largest obtained with any stimulus to date. These in vitro observations may provide an important link to findings with experimental models of glomerular disease. which directly associated the induction of mesangial proliferation with the glomerular deposition of terminal complement A

Effects

of

Incorporation

0

Control

0

C5b-8

A

C5b-9

C Sb-8 of AA

and Into

C Sb-9

on

8

Effects

MESANGIAL

CELLS

components (32). Thus, our findings suggest that terminal complement-mediated stimulation of MC IL 1 release may mediate the mesangial cellular proliferation characteristic of many forms of glomerular disease. C5b-9 proved to be an effective stimulant of mesangial prostanoid release. When assessed at later time points, PGE was the predominant prostanoid species released. PGE and prostacyclin secretion could also be induced by exposure to C5b-8, indicating that a PG secretory response could occur in the absence of the complete terminal complex. C5b-9 previously has been found to initiate arachidonate and prostanoid release from Ehrlich ascites cells, macrophages, and platelets (17, 22, 33). suggesting that this may represent a common mechanism whereby complement modulates inflammatory processes. Prostacyclin secretion was induced by C5b-9 very rapidly and ceased within 1 hr. Although enhanced PGE release was observed at 1 hr. the rate of secretion increased greatly at 6 hr after complement stimulation. This major difference in the kinetic patterns of prostacyclin and PGE release suggests that separate control mechanisms may be involved. The rapid phase of prostanoid secretion in response to C5b-9 is most consistent with the transient activation of phospholipases following development of an intramembranous complement complex. Phospholipase AZ activation may be mediated through the development of structural membrane alterations or by a rapid, complement-mediated calcium influx, as has been observed with polymorphonuclear leukocytes (34, 35). On the other hand, the sustained PGE release may be more indirect and mediated through complement-stimulated mesangial IL 1. We observed recently4 that IL 1 acts rather specifically to induce mesangial secretion of PGE. The kinetic pattern of IL 1 release in response to complement, which peaks at 6 hr. corresponds to the period of enhanced PGE secretion by the MC. The direct testing of this hypothesis awaits the development of a specific anti-rat-IL 1 antibody. The experiments concerning PL metabolism provide more insight into the possible mechanisms of C5b-9 action on MC. AA was rapidly released from the PL PC, PI, and PE after complement treatment. This coordinate

of C 5b -8

lncorporatlon

PC

OF

of

and

AA

Into

C5b

-9 on

C

PI

1.2

Effects

0

T 3 0 Q

of

hcorporatlon

C5b-8

and

of AA into

C5b-9

on

PE

Control

0

C5b-8

A

C5b-9

.F 0.6

I 15

I 60

1 120

Figure

10.

Effects

of C5b-8

and C5b-9

$0

0 Time

Time (minutes)

on AA incorporation

( minutes)

into PC [A). PI [B). and PE (C). Experimental

1I

details

$0 Time

are as described

(minutes)

for Figure

8.

COMPLEMENT ACTIVATION OF MESANGIAL CELLS

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phase of antiglomerular basement membrane nephritis.Kldney Int. release suggests that a common mechanism, the activa27:643. tion of phospholipase A2. is involved, resulting in a large ndre,and A. Martinez7. Koffler. D., G. Biesecker, B. Noble. 0. A. A Hernandez. 1983. Localization of the membrane attack complex in increase in the size of the intracellular free fatty acid experimental immune complex glomerulonephritis. J. Exp. Med. pool. This increased substrate is thenpresumably avail157:1885. 8. Adler, S..P.J. Baker, P. h i t z l , a n dW. G. Couser. 1984. Detection able for the PG synthetic pathways. In addition to the of terminal complement components in experimental glomerular rapid release of arachidonate from phospholipid pools, injury. Kfdney Int. 26:830. complement stimulated the incorporation of exogenously 9. Perkinson, D.T., P. J. Baker. W.G. Couser, R. J. Johnson, and S. Adler. 1985. Membrane attack complex deposition in experimental supplied arachidonate back into cellular PL. The fatty glomerular injury. Am. J . Pathol. 120:121. acid moieties of PL in cellular membranes are in a dy- 10. Biesecker. G.. S. Katz, and D. Koffler. 1981. Renal localization of namic equilibrium due to a n enzymatic deacylation-reathemembraneattack complex in systemic lupuserythematosus nephritis. J. Exp. Med. 154: 1779. cylation cycle. The present findings indicate that, after 11. Fa&, R. J., A. P.Dalmasso, Y. Kim, C. H. Tsai. J. I. Scheinman. H. complement stimulation, both aspects of this cycle are Gewurz, and A. F. Michael. 1983. Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal active. The increase in arachidonate incorporation into antibody and immunohistochemical localization in renal disease. J . the PL may be due to increased availability of substrate Clln. Invest. 72:560. lysophosphatide (a product of phospholipase A2 activa- 12. Lovett, D. H.. J. L. Ryan, and R.B. Sterzel. 1983. A thymocyte activating factor derived from glomerular mesangial cells. J . Immution) or due to a stimulation of the activity of the key nol. 130: 1 796. reacylation enzyme, lysophosphatide acyltransferase 13. Lovett, D. H., M. Szamel, J. L. Ryan, R. B. Sterzel, D. Gemsa. and (E.C.2.3.1.23). The persistent activity of the mesangial K. Resch. 1986. Interleukin1 and the glomerular mesangium. I. Purification and characterization of a mesangial cell-derived autoacyltransferase enzyme after complement treatment congrowth factor. J . Immunol. 136:3700. trasts with our previous findings with complement-stim- 14. Lovett, D. H..R.B. Sterzel, M. Kashgarian, and J. L. Ryan. 1982. ulated platelets (22).In these experiments, a loss of acylNeutral proteinase activity produced in vitro by cells of the glomerular mesangium. Kfdney Int. 23:342. transferase activitywas observed, correlatingwitha 15 Yamamoto. K.. and H. Gewurz. 1978. Thecomplex of C5b and C6: massive release of thromboxane due to enhanced subisolation, characterization, and identification of a modified form of C5b consisting of three polypeptide chains. J . Immunol. 120:2008. strate availability. Complement-induced platelet aggreC.H.,G.H. Wirtz, L. Renfer, H.D. Gresham, and B. F. gation, with loss of functionalintegrity (36),may be 16 Hammer, Tack. 1981. Large scale isolation of functionally active components responsible for this absence of acyltransferase activity, of the human complement system. J . Blol. Chem. 56:3995. a n event which does not occur with complement-treated 17. Haensch, G. M., M. Seitz, G. Martinotti, M. Betz, E. W.Rauterberg, and D. Gemsa. 1984. Macrophages release arachidonic acid, prosMC. The maintenance of mesangial acyltransferase actaglandin E and thromboxane in response to late complement comtivity after complement attack may be related to the ponents. J . Immunol. 133:2145. general complement resistance of nucleated cells alluded 18. Grimm. W.,M. Seitz. H. Kirchner, andD. Gemsa. 1978. Prostaglandin synthesis in spleen cell cultures of mice injected with Coryneto previously. bacterium paruum. Cell. Irnmunol. 40:419. In summary, purified components of the terminal MAC 19. Gemsa, D., W. Kramer. M. Brenner. G. Till, and K. Resch. 1980. Induction of prostaglandin E release from macrophages by colchiof complement stimulate the release of prostanoids and cine. J . Immunol. 124:376. of a n autocrine mesangial cell growth factor, IL 1 . These 20. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of events are coupled with a n enhanced turnover of mesprotein dye binding. Anal. Blochem. 72:248. angial membranePL fatty acids. Together,these findings 21. Goppelt. M.. and K. Resch. 1984. Densitometric quantitation of are characteristicof cellular activation, andsuggest that individual phospholiplds from natural sources separated by onedimensional thin-layer chromatography. Anal. Blochem. 140:152. other phenotypic properties of the MC may also be al22. Haensch, 0. M.. D. Gemsa, and K. Resch. 1985. Induction of prostered. Complement-mediated activation of a n intrinsic tanoid synthesis in humanplatelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of glomerular cell type, with resultant release of biologically active molecules, provides further evidence for the sig- 23. the reacylation of liberated arachidonic acid.J. Immunol. 235:1320. Koski. C. E., L. E. Ramm. C.H. Hammer, M.M. Mayer, and M. L. nificant role of terminal complement components in the Shin. 1983. Cytolysis of nucleated cells by complement: cell death displaysmulti-hitcharacteristics. Proc.Nut(. Acad. Scl. USA mediation of immunologic glomerular disease. 24.

Acknowledgments. The authors thankCarol Edwards for preparation of the manuscript, Dr. Elisha Atkins for his helpful discussion of the data, and the Alexander von Humboldt Stiftung for the extended support of this project.

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