Cell Cycle-dependent Interleukin 1 Gene Expression by CulturedGlomerular Mesangial CellsDavid H. Lovett* and Alf Larsent*Medical Service, Veterans Administration Medical Center-University of California, San Francisco, San Francisco, California 94121;and tDepartment of Molecular Biology, Immunex Corporation, Seattle, Washington 98101

Abstract

Glomerular mesangial cell (MC)-derived IL-1 may be an im-portant factor in the development of the hypercellularity andsclerosis characteristic of many forms of glomerulonephritis.To define the regulation of IL-I synthesis by human MC,Northern blot analyses were performed using specific probesfor monocytic IL-1 a and 8 mRNA. Proliferating MCex-pressed mRNAfor both IL-1 a and ft, whereas nonproliferat-ing MCcontained no detectable IL-1 mRNA. SynchronizedMCexpressed IL-i a and ft mRNAwithin 2 h of stimulationwith serum. This serum effect could be reproduced with plate-let-derived growth factor and epidermal growth factor. Im-mune precipitations of 35S-methionine-labeled cells indicatethat the mesangial IL-1 is synthesized as a 33-kD precursorprotein with a p1 of 7.2. Extracellular mesangial IL-1 has a plof 7.0 and molecular weight of 17 kD, consistent with its iden-tification as IL- fl. Cellular proliferation in glomerular dis-ease may be driven in part by peptide growth factor-mediatedinduction of mesangial IL-1 gene expression and protein syn-thesis.

Introduction

IL-1 represents a family of proteins that have a central func-tion in the development of immune and inflammatory pro-cesses. Originally viewed as a product solely of activatedmonocytes, it is now generally recognized that molecules dis-playing IL-1 activity are produced by many discrete cellulartypes. These include glial cells, endothelial cells, keratinocytes,B cells, and glomerular mesangial cells (MC),' among others(1-4). Biochemical characterization of the monocyte-derivedIL- 1 has demonstrated the existence of at least two distinctgene products, termed IL-I a and IL-1 ,B (5-8). ExtracellularIL- 1 has a molecular mass of 17 kD and displays charge heter-ogeneity, with prominent species at pI 7.0 and 5.0. It has beensuggested that the pI 5.0 and pl 7.0 forms of extracellular IL- Icorrespond to the IL- 1 a and IL- 1 , sequences, respectively(9). Both forms of monocyte IL-I appear to be translated as

Address correspondence to Dr. Lovett, 11I J Medical Center, Univer-sity of California Medical Service, San Francisco VAMC, 4150 Clem-ent St., San Francisco, CA 94121.

Receivedfor publication 20 July 1987 and in revisedform 1I De-cember 1987.

1. Abbreviations used in this paper: EGF, epidermal growth factor;HMC, human MC; IEF, isoelectric focusing; IF, immunofluorescence;IGF- 1, insulin-like growth factor- 1; ITS, insulin, transferrin, selenium;MC, mesangial cell; PDGF, platelet-derived growth factor.

The Journal of Clinical Investigation, Inc.Volume 82, July 1988, 115-122

31-33-kD precursor proteins (9, 10). The IL- 1 f species repre-sents the principal form of IL- 1 mRNAsynthesized by endo-toxin-stimulated human monocytes. In contrast, human kera-tinocyte-derived IL- 1 mRNAwas found to consist primarily ofthe IL- I a species, indicating that the type of IL- 1 synthesizedmay be cell specific (1 1).

The IL- 1 activity produced by cultured rat glomerular MChas been characterized at both the functional and biochemicallevels. MCIL- 1 stimulates IL-2 secretion from splenic lym-phocytes and is active as an endogenous pyrogen (4, 12). Mostsignificant may be the observation that purified MCIL- I wasfound to act as an autogrowth factor for cultured MC(13).This may be of potential relevance to the development of thehypercellularity seen in many glomerular diseases. Purified ratMCIL- 1 has a molecular mass of 14 kD and displays chargeheterogeneity, with pls of 7.2, 5.0, and 4.45 (13). Because ofthe lack of cross-reactivity of available antibodies with the ratIL-1, it is not presently possible to characterize the variousisoelectric species as IL- 1 a or ,B analogues.

The responsiveness of MCto various peptide growth fac-tors, including platelet-derived growth factor (PDGF) and epi-dermal growth factor (EGF), has been recently documented(14, 15). In our own studies a relationship between IL-l secre-tion and MCproliferation, a process driven by peptide growthfactors, was observed (13). However, the precise kinetic pat-terns of MCIL- 1 production and the identification of thepathologically relevant mediators of this process remainedlargely unexplored. Given the recent availability of specificreagents for the evaluation of the human monocytic IL- 1 sys-tem, we elected to more closely define the relationships be-tween MCactivation by peptide growth factors, MCIL- 1 geneexpression, and IL-1 secretion.

Methods

Reagents. EGF, receptor grade, and HPLC-purified PDGFwere ob-tained from Collaborative Research, Inc. (Waltham, MA). ITS, con-taining 5 Ag/ml human transferrin, 1 X 10-6 Minsulin, and 5 ng/mlselenous acid (final concentrations in medium) was obtained from thesame source. Goat anti-human PDGFIgG was the generous gift of E.Raines and R. Ross, Dept. of Pathology, University of Washington.Before use, the various growth factors and antibodies were absorbedwith polymixin B-Sepharose (100 Al packed beads/ml component) andwere free of detectable (< 0.1 ng/ml) endotoxin contamination whenassessed by an amoebocyte lysate assay (the generous gift of J. L. Ryan,Yale University, NewHaven, CT). All other media components werescreened in a similar manner.

Establishment of human MC(HMC) cultures. Humankidney wasobtained at the time of elective nephrectomy from patients with var-ious forms of well-circumscribed renal neoplasia, and primary culturesof HMCwere established from collagenase-treated glomeruli as re-cently reported in detail (16). The growth medium consisted of RPMI1640 supplemented with 20% heat-inactivated FCS (Gibco, GrandIsland, NY), 50 U/ml penicillin, 50 Ag/ml streptomycin, and 300

Mesangial Cell Interleukin I Gene Expression 115

Mg/ml glutamine. ITS was routinely added to all culture media. Cellu-lar outgrowths appeared within 5 d and consisted of interwoven,straplike cells. Small clusters of these cells (approximately five) werepicked out, replated, and grown to confluency. The cultures were sub-sequently rapidly expanded and uniform populations were frozen inliquid nitrogen at the fourth to sixth passages. For experiments, thecells were thawed, plated in growth medium, grown to subconfluency,and used as detailed below.

The cells were characterized as intrinsic mesangial using previouslyreported criteria (16-18). Immunofluorescence (IF) staining revealedprominent intracellular myosin fibrils arranged along the long axis ofthe cells. IF was negative for factor VIII, common leukocyte antigen,and Ia antigen. Confluent cultures were multilayered, and there was nomorphologic evidence for the presence of either glomerular endothelialor epithelial cells, which grow in monolayers of polygonal cells (19).Studies of protein synthetic profiles using [3H]proline and [35S]-methionine labeling revealed the secretion of fibronectin, thrombo-spondin, and collagens type I, III, IV, and V (submitted for publica-tion). For light microscopic evaluation, the HMCwere washed threetimes with PBSand fixed with 3.7% paraformaldehyde in PBS, 1 mMMgCl2, and 0.1 mMCaCI2 for 20 min at room temperature. Afterfixation the cells were evaluated by light microscopy after Giemsastaining (Fig. 1). HMCwere prepared for electron microscopy usingfixation and embedding procedures previously reported in detail (20).Ultrathin sections were cut, mounted, and stained with 2% aqueousuranyl acetate and lead citrate before examination with a JEOL elec-tron microscope (Fig. 2).

Cell synchronization. For synchronization studies the HMCwerewashed twice with warm PBS and incubated for 48-72 h in RPMI1640 supplemented with ITS and 0.5% BSA (rest medium). Supple-mental insulin and transferrin were found to be required for the main-tenance of cell viability under these serum-free conditions (21), andwere used without modification for these experiments. Previous stud-ies have demonstrated that under these culture conditions MCcease tosecrete significant quantities of IL-I after 24 h (13). For stimulationstudies the rest medium was removed and various combinations ofpeptide growth factors or serum were added as detailed below.

Preparation ofHMCRNA/NAorthern blotting. HMCcell layers werewashed twice with cold PBS, released by gentle scraping, and centri-fuged at 400 g for 10 min. Total RNAwas extracted from the pellets bythe guanidine isothiocyanate/cesium chloride method (22). TotalRNAfrom endotoxin-stimulated human peripheral blood monocyteswas prepared in the same manner (5). All RNApreparations used werefree of degradation.

For Northern blot analysis, 5 uig total RNAper lane was electro-phoresed in 1.5% agarose gels containing formaldehyde and trans-ferred to nitrocellulose filters. Blots were hybridized with [32P]RNAprobes synthesized by SP 6 polymerase as previously reported (5). TheIL I a probe was transcribed from a gemini (Promega-Biotec., Madi-son, WI) subclone of the 660-bp Hind III-Hinc II fragment of p10 A,and the IL- I probe was transcribed from a gemini subclone of the570-bp Sac I-Pvu II fragment from p IL-1-14 (5). The histone H2bprobe was the generous gift of P. Challoner (Fred Hutchinson CancerResearch Center, Seattle, WA) and was derived from a 2.3-kb HindBIII-Bam HI fragment as reported (23). After hybridization for 16 h at630C in Stark's buffer, the blots were washed with 0.1 X standardsaline citrate ard autoradiographed at -70'C with intensifyingscreens. The positions of ribosomal 28 and 18 S RNAs were deter-mined by visualization of unbaked blots with short wave UVlight. TheNorthern blots presented are representative of experiments performedusing several independently derived lines of HMC.

IL-I assay. The levels of biologically active IL-l in the HMCsu-pernates were quantitated in a conventional thymocyte co-mitogenesisassay. In brief, 1.5 X 16 thymocytes from 8-10-wk-old C3H/HeJ mice(Charles River Breeding Laboratories, Wilmington, MA) were cul-tured in 200 Ml RPMI 1640 supplemented with 5% FCS, 50 U/mlpenicillin, 50 Mg/ml streptomycin, 2 mMglutamine, 1 X I0- M fl-mercaptoethanol, and 0.5 ug/ml concanavalin A. The cultures con-

tained serial dilutions of test materials or serial dilutions of purifiedhuman recombinant IL- I # as a standard. The cultures were pulsed at48 h with 1 MCi [3H]thymidine/well (2 Ci/mmol, New England Nu-clear, Boston, MA) and terminated 24 h later using an automated cellharvester. IL- I activity was calculated by probit analysis, in which thestandard recombinant IL- I j at a concentration of 1 U/ml induced a50% maximal thymocyte proliferative response under the conditionsused. The protein content of trichloroacetic acid-precipitated celllayers was determined by the method of Bradford (24) using BSAas astandard.

Biosynthetic labeling/immune precipitation. Subconfluent HMCcultures were placed in rest medium for 72 h as detailed above. There-after, the rest medium was removed and the cells were incubated withmethionine-deficient' DMEfor 60 min. This medium was then re-moved and replaced with fresh methionine-deficient DMEcontaining100 MCi/ml [35S]methionine (1,350 Ci/mmol, NewEngland Nuclear),PDGFat 5 ng/ml, EGFat 100 ng/ml, and ITS. After culture for 6 h,the supernates were removed, centrifuged at 400 g for 10 min, supple-mented with protease inhibitors (5 mMEDTA, 0.2 mMphenylmeth-ylsulfonyl fluoride, and 2 AMpepstatin), and frozen at -70'C. TheHMCcell layers were washed twice with cold PBS, scraped into mi-crofuge tubes, and centrifuged at 1,000 g for 5 min. The cell pelletswere then lysed in an equal volume of two times lysis buffer consistingof (final concentrations) 2%NP-40, 0.15 MNaCI, 10 mMNaH2PO4,10 mMNa2HPO4, 0.68 Msucrose, pH 7.2, and protease inhibitors asgiven above. Cell lysates were incubated on ice for 30 min, sonicatedbriefly, and centrifuged at 10,000 g for 15 min. For immune precipita-tion of radiolabeled intracellular IL-1, the cell lysates were preclearedby incubation at 4°C overnight with 10% vol/vol pansorbin (Calbio-chem-Behring Corp., La Jolla, CA). Cleared lysates were then incu-bated for 18 h at 4°C overnight with a 1:20 final dilution of rabbitpolyclonal anti-human IL-1 IgG (Genzyme, Boston, MA). This anti-body preparation recognized both the pI 7.0 and pI 5.0 forms of mono-cyte IL- I in experiments utilizing either immune precipitation or im-munoblotting (not shown). Nonimmune rabbit IgG was used as anegative control. After incubation, 10% pansorbin was added and thebound immune complexes were recovered by centrifugation. The pel-leted bacteria were washed five times in 100-fold excess volumes ofbuffer containing 0.5% NP-40, 0.45 MNaCl, and 0.05 MTris/HCl, pH8.3. The pellets were finally resuspended in isoelectric focusing (IEF)sample buffer (9.3 Murea, 5 mMK2CO3, 2%NP-40, 2%3-10 ampho-lines, and 30 mMdithiothreitol). After a 2-h incubation at 4°C, thesamples were centrifuged and analyzed by two-dimensional electro-phoresis. Immune precipitation of radiolabeled IL-i present in theculture supernates was performed in an identical fashion.

Electrophoretic methods. The proteins recovered by immune pre-cipitation were analyzed by two-dimensional electrophoresis and auto-radiography. IEF was performed on 0.4-mm ultrathin polyacrylamideslabs'according to Goldsmith et al. (25), using 1.4% 4-6 ampholinesand 0.6% 3-10 ampholines (LKB, Bromma, Sweden). Focused pro-teins were separated in the second dimension on 12.5% SDS-PAGEdiscontinuous gels precisely according to O'Farrell (26). After electro-phoresis the gels were fixed in 10%TCA/30% ethanol for 45 min. Fixedgels were washed twice in 5% TCA/30% ethanol, soaked in Enhance(New England Nuclear), and dried under heat and vacuum. Autoradi-ography was performed at -70°C using preflashed Kodak X-O-Matfilm with intensifying screens. pI were determined from the migrationpatterns of stained standard proteins (IEF calibration kit, pI 3 to 10range; Pharmacia Fine Chemicals, Piscataway, NJ). Phosphorylase b(94 kD), bovine serum albumin (67 kD), ovalbumin (45 kD), trypsininhibitor (20 kD), and lactalbumin (14 kD) were used as molecularmass standards for SDS-PAGE.

Results

The cells used in these studies were homogeneous and repre-sentative of the intrinsic glomerular MC, which resembles in

116 D. H. Lovett and A. Larsen

Figure 1. Light micrograph of culturedHMC. Giemsa stain. Cells have stellate orfusiform shape and grow in interwovenbundles. X 600.

many respects the vascular smooth muscle cell (27). As seen inFig. 1, the cultured HMCare straplike in shape, with a promi-nent intracellular fibrillar component. As with rat MC, cul-tured HMCtend to grow in multilayers and produce abundantextracellular matrix. These features are also appreciated by

transmission electron microscopy (Fig. 2). The cells displayperipherally located heterochromatin and have an elongatedmorphology. Prominent rough endoplasmic reticulum andGolgi apparatus are present. In areas, large accumulations ofextracellular matrix were noted. These features are character-

giv

44.

Figure 2. Electron micrograph of cultured HMC. Cells are present in multilayers with an axially elongated morphology. HMCcontain periph-erally condensed nuclear chromatin, prominent Golgi apparatus, and abundant rough endoplasmic reticulum. X 8,400.

Mesangial Cell Interleukin I Gene Expression 117

A B

28S-_

S

1 2 3

.~~~~

1 2 3Figure 3. Northern blot analysis of RNAsamples extracted from en-dotoxin-stimulated monocytes (lanes Al and BJ), noncycling HMC(lanes A2 and B2), and cycling HMC(lanes A3 and B3). Aliquots oftotal RNA(5 Ag) were electrophoresed, transferred, and analyzedwith 32P-labeled probes specific for human monocyte IL- l a (A) andIl-1 ,B mRNA(B). The positions of the 28 S and 18 S ribosomal sub-units are given by the arrow markers.

istic of the intrinsic MCand exclude contamination of thecultures by endothelial or monocytic cells.

Northern blot analysis of total RNAextracted from lipo-

A

polysaccharide-stimulated monocytes, actively cycling HMC(maintained in medium supplemented with 20% FCS), andnoncycling HMCwas performed (Fig. 3). As expected, stimu-lated human monocytes contain large amounts of mRNAforboth IL-l a and ,B species (lanes Al and BJ). ProliferatingHMC(lanes A3 and B3) were also found to contain mRNAofboth IL- 1 species, but in lesser abundance than monocytes.Densitometry of autoradiograms indicated that IL-l B mRNAwas 1I0-fold more abundant than IL-l a mRNAin the cy-cling HMCpopulation. In contrast to the results obtained withproliferating HMC, noncycling HMChad little or no detect-able IL- l mRNAof either type (Fig. 3, lanes A2 and B2). Thus,these experiments demonstrated that HMCcan expressmRNAfor both IL-l species, and that this expression is de-pendent upon the proliferative status of the cell population.

To more closely examine the relationship of cellular prolif-eration to the expression of IL- 1 mRNA,cultured HMCweresynchronized into a rest state in medium deficient of serum,but containing BSA, insulin, and transferrin. Experimentalgroups were induced to reenter the cell cycle by exposure tocomplete medium containing 20% FCS. Controls were givenfresh medium devoid of serum. As seen in Fig. 4 A, theseresting cells do not express detectable quantities of IL-1mRNA,confirming the findings shown above in Fig. 3. Whenresting cells were exposed to 20% serum (Fig. 4 B), there ap-peared a time-dependent expression of both IL-1 a and ,3mRNA. 2 h after serum exposure both IL-l mRNAspecieswere detectable; however, the subsequent kinetic patterns ofIL-1 a and # mRNAexpression were different. As notedabove, the IL-1 , mRNAspecies is more abundant that theIL- 1 a species in this serum-stimulated cell population.

B C

..

.5 1 2 4 6 12 24 .5 1 2 4 6 12 24 .5 I 2 4

T IME 'h,Figure 4. Kinetic pattern of HMCIL- I mRNAexpression. HMCwere synchronized into a rest state using serum-free medium supple-mented with BSA, insulin, and transferrin. Thereafter, the HMCwere incubated with either fresh rest medium (A), with medium sup-

plemented with 20% FCS (B), or with rest medium supplemented

with 5 ng/ml PDGF(C). RNAwas extracted at the given time inter-vals and Northern blot analysis for expression of II- 1 a and Il-1 I,mRNAwas performed. In B, the upper hybridization band repre-

sents Il- I a mRNA,while the lower band represents II-1 B mRNA.

118 D. H. Lovett and A. Larsen

6 12 24

18s-.*.

While serum stimulation of resting HMCallowed furtherdefinition of the cell cycle-dependent nature of IL-1 gene ex-pression by HMC,we wished to define a minimal combinationof relevant peptide growth factors that could reproduce thisserum effect. Previous studies have demonstrated that PDGFis one of the major mesangial mitogens (14, 18). In addition,the mitogenic effect of EGFon MChas been recently reported(15). Therefore, the effects of various combinations of PDGFand EGF on IL-1 mRNAexpression were examined usingmitogenically relevant concentrations (references 14 and 15,and unpublished observations). When resting HMCwere ex-posed to 5 ng/ml purified PDGF, only minimally detectablelevels of IL- 1 mRNAof either type were noted (Fig. 4 C).EGF, at 100 ng/ml, also failed to induce IL-1 mRNAexpres-sion (Fig. 5 A). Whenthese same concentrations of PDGFandEGFwere used together, a time-dependent expression of IL-lmRNAfor both a and # were observed (Fig. 5 B). As withserum stimulation, the purified peptide growth factors appearto induce significantly more IL-1 ft than IL-1 a mRNA. Inaddition, the kinetic patterns of mRNAexpression for thesetwo species do not appear to be identical. As a control for thespecificity of PDGFaction in IL-l gene expression, restingHMCwere treated with the same concentrations of PDGFandEGFin the presence of 50 Ag/ml goat anti-PDGF IgG (Fig. 5C). Inclusion of specific anti-PDGF antibodies reduced theexpression of IL- 1 mRNAof either type to undetectable levels.

Expression of mRNAfor the S-phase-specific histone H2b(23) was used as a marker for peptide growth factor inductionof cellular proliferation and to localize IL- I mRNAexpression

A

to a specific portion of the cell cycle (Fig. 6). Resting HMCexposed to either PDGF(Fig. 6 A) or EGF (Fig. 6 B) aloneexpressed low, constant levels of H2b mRNA.After combinedexposure to PDGFand EGF(Fig. 6 C), enhanced H2b mRNAexpression was noted at 12 and 24 h, indicating onset of cellu-lar proliferation. This pattern of histone mRNAexpressionhelps to localize the initiation period of IL-l mRNAexpres-sion to the GI phase of the cell cycle. The pattern of histoneH2b mRNAexpression, which does not mirror IL-1 mRNAexpression, also demonstrates that the increase in IL- 1 mRNAlevels observed is not simply due to generalized increases inmRNAtranscription rates.

To correlate IL-1 mRNAexpression with synthesis of afunctional IL- I molecule(s), the amounts of biologically activematerial in culture supernates were quantified. For these ex-periments, resting HMCwere stimulated with PDGF(5ng/ml) and EGF (100 ng/ml) in the presence of insulin andtransferrin. Media conditioned for discrete time intervals aftergrowth factor stimulation were harvested and biologically ac-tive IL-l quantified in the standard murine thymocyte assay.These results are shown in Fig. 7. Biologically active IL- I wasreleased in the culture medium during a limited time period of2-6 h after growth factor stimulation.

The biochemical properties of the HMCIL-l activity in-duced by growth factor stimulation were assessed by immuneprecipitation and two-dimensional electrophoretic analysis of[35S]methionine-labeled material. For these experiments apolyclonal anti-human monocyte IL-l preparation was usedto recover the intracellular and extracellular forms- of HMC

B C

0 .5 I 2 4 6 12 24 0 .5 l 2 4 6 12 24 0 2 4 6

TIME h)Figure 5. Kinetic pattern of HMCIL- I mRNAexpression (continued from Fig. 4). The HMCwere synchronized into a rest state as detailedabove. The HMCwere then incubated with fresh rest medium supplemented with 100 ng/ml EGF(A) or with medium supplemented with 5

ng/ml PDGFand 100 ng/ml EGF(B). In C, the HMCwere treated with 100 ng/ml EGF, 5 ng/ml PDGF, and 50 Ag/ml goat anti-PDGF IgG.

Mesangial Cell Interleukin 1 Gene Expression 119

18 S..w

A94-

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45-

B20_

14_

. #~~e IVI:,..i ...

C0 .5 1 2 4 6 12 24

KD

*4::: ::.

7 6 5 4a I I I I

pH

TIME h)Figure 6. Kinetic pattern of S-phase specific histone H2b mRNAex-

pression. (A) Resting cells treated with 5 ng/ml PDGF; (B) restingcells treated with 100 ng/ml EGF; (C) resting cells treated with 100ng/ml EGFand 5 ng/ml PDGF. The arrow in Cdenotes the onset ofenhanced H2b transcription, which corresponds to the initiation ofcellular DNAsynthesis.

IL-i. After 6 h of stimulation with PDGF, EGF insulin, andtransferrin, a single 33-kD protein with a pI of 7.2 was detect-able in immune precipitates from cellular lysates (Fig. 8,

50t

CDO /O 30

-J7Co~_ l

10.

C D E

HARVESTINTERVALS

Figure 7. Temporal pattern of HMCIL- I secretion stimulated bypeptide growth factors. Resting HMCwere treated with 100 ng/mlEGFand 5 ng/ml PDGFas above. HMCcultures were allowed tocondition the culture media for discrete time intervals. The levels ofIL- I present in the conditioned media were determined in the thy-mocyte assay. Data given as mean±SD (n = 4). Interval A, 0-0.5 h;interval B, 0.5-1 h; interval C, 1-2 h; interval D, 2-4 h; interval E,4-6 h; interval F, 6-12 h; and interval G, 12-24 h.

94-

67

45-

30-

20_

14-

KD

(.)

pH

Figure 8. Immune precipitation and two-dimensional electrophoreticanalysis of "5S-methionine-labeled HMCIL- 1. Resting cells weretreated for 6 h with 100 ng/ml EGFand 5 ng/ml PDGFin the pres-ence of 100 uCi/ml "5S-methionine. Radiolabeled IL-I was specifi-cally immune precipitated from cell layers (upper panel) or the cul-ture supernates (lower panel). The immune precipitates were sepa-rated by two-dimensional electrophoresis as outlined in Methods,followed by autoradiography.

upper). From the culture medium a single labeled protein witha molecular mass of 17 kD and pI of 7.0 was recovered (Fig. 8,lower).

DiscussionUsing well-characterized human glomerular MC, the expres-sion of IL- I mRNAand IL- I protein synthesis was examined.Randomly proliferating HMC, in contrast to resting cells, ex-pressed mRNAfor both the IL- 1 a and # species. MesangialIL- 1 mRNAelectrophoretically co-migrated with monocyteIL- 1 mRNA,but appears to be less abundant. Serum or pep-tide growth factor stimulation of resting HMCresulted in a

120 D. H. Lovett and A. Larsen

III

limited period of expression of both IL-I a and a mRNA. Ineach case, IL-i I3 mRNAwas the predominant transcript ob-served. Biologically active IL-I was secreted into the culturemedium during the period of maximal IL-I mRNAexpres-sion. IL- I # mRNAexpression persisted somewhat beyond theperiod of IL- I protein secretion, suggesting that posttranscrip-tional regulatory events are involved. PGEhas been recentlynoted to posttranscriptionally inhibit monocyte IL- I secretionby raising intracellular cAMPlevels (28). IL- 1 is a potent andrapid stimulant of MCPGEsecretion, (29), and it is likely thata similar form of regulation occurs in this cell type.

Immune precipitation of cellular lysates after 6 h of peptidegrowth factor stimulation in the presence of [35S]methionineyielded a single-labeled protein with a molecular mass of 33kD and pI of 7.2. From the culture supernates of these growthfactor-stimulated cells, a single-labeled protein with a molecu-lar mass of 17 kD and pI of 7.0 was recovered. The 33-kDintracellular protein presumably represents the same precursorIL- 1 protein recently described in stimulated monocytes (9,10, 30). The 1 7-kD extracellular mesangial IL- I protein closelyresembles the most prominent secretory form of IL- I derivedfrom endotoxin-stimulated monocytes and presumably repre-sents the IL-1 (3 translation product (9). Stimulation of MCwith the terminal complement components, C5b-9, also ledto the release of IL- I with a pI of 7.0, (31), suggesting that thispl species represents the major secretory form of mesan-gial IL-1.

The combination of a fixed concentration of PDGFandEGFin a maintenance medium containing insulin and trans-ferrin was sufficient to induce HMCIL- 1 gene expression andprotein secretion. It is very probable that other combinationsof peptide growth factors, or varying concentrations of theseindividual factors, may induce IL-I gene expression by MC.These issues are presently under investigation. The require-ment for transferrin to maintain HMCviability is common tomost culture systems using serum-free media and does notsuggest a specific role for transferrin in the regulation of IL-Igene expression. MCappear to lack insulin receptors; how-ever, specific insulin-like growth factor-I (IGF-I) receptorshave been identified (32). At the supraphysiologic concentra-tions of insulin used in these studies (1 X I0O M), insulin canbind to the lower affinity IGF-I receptors. Some IGF-I activityappears to be required to maintain HMCviability, as cultureswithout this concentration of supplemental insulin quicklydied. As with transferrin, the requirement for insulin in theinduction of IL- I gene expression may be more related to thenecessary maintenance of cellular viability than to any specificIL-I effect.

A comparison of IL-I gene expression and protein secre-tion by monocytes and other cells may provide some insightsinto the regulation of this factor by specific cellular types. Inendotoxin-stimulated monocytes, IL-i I #mRNAlevels weredetected as early as 1 h (30). IL-i I mRNAlevels were maxi-mal by 6 h, but remained elevated for at least 16-24 h afterendotoxin stimulation (5, 30). IL- I a mRNAwas not detectedat early time points (i.e., < 6 h), but could be demonstratedafter 24 h of endotoxin stimulation (5, 30). The kinetic patternof monocyte IL-I secretion generally parallels IL-I mRNAexpression and is of prolonged duration (30, 33). While theprecise kinetic pattern was not identified, human endothelialcells were found to express exclusively IL- I , mRNAafter 18 hof endotoxin stimulation (34). IL- I a mRNAwas observed in

endothelial cells only after combined treatment with endo-toxin and cycloheximide (34). A very similar pattern of IL-ImRNAexpression was observed in aortic vascular smoothmuscle cells (35). The biochemical properties of secreted endo-thelial and vascular smooth muscle cell IL-I have not beenclearly defined, and it is presently uncertain whether theseproducts are identical with the monocytic counterparts. Thekinetic patterns of HMCIL- I mRNAexpression and proteinsecretion observed in these studies therefore appear to be dis-tinctive. Our findings suggest that these events may be regu-lated in a cell-specific manner or that the nature of the stimu-lus (endotoxin vs. growth factors) may be of importance.

The involvement of the intrinsic MCis evident in mostforms of glomerular disease associated with the developmentof sclerosis (36). The linkage between MCproliferation andmatrix expansion observed at the histologic level has served asthe basis for in vitro studies examining the determinants ofMCgrowth. Recently, a relationship between cellular prolifer-ation and augmented synthesis of mesangial matrix was dem-onstrated in long-term MCcultures (20). Therefore, determi-nation of the relevant inflammatory factors that drive thisproliferative process is an important experimental goal. Thesestudies have identified PDGFand EGFas stimulants of mes-angial IL-I secretion. In glomeruli, several potential cellularsources for these growth factors are present. Platelet activationoccurs frequently in glomerular inflammation and may resultin the release of both PDGFand EGF (37-39). In addition,endothelial cells secrete a number of mitogens, including mol-ecules with PDGF-like properties (40). Local release of PDGFand EGFfrom these sources during glomerular inflammationmay represent a commonmechanism whereby mesangial pro-liferation and matrix expansion are initiated.

Acknowledaments

The authors thank L. Chen for technical assistance and S. Gillis for acritical review of the manuscript.

This research was supported in part by grants from the VeteransAdministration and the Northwest Kidney Foundation.

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122 D. H. Lovett and A. Larsen