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Originally published In Press as doi:10.1074/jbc.M200419200 on April 30, 2002

J. Biol. Chem., Vol. 277, Issue 30, 27535-27544, July 26, 2002
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Opposite Regulation of Type II and III Receptors for Immunoglobulin G in Mouse Glomerular Mesangial Cells and in the Induction of Anti-glomerular Basement Membrane (GBM) Nephritis*

Heinfried H. RadekeDagger §, Iska Janssen-Graalfs||, Eveline N. SowaDagger ||, Nelli Chouchakova, Julia Skokowa, Fabian Löscher, Reinhold E. Schmidt, Peter Heeringa**, and J. Engelbert GessnerDaggerDagger

From the  Department of Clinical Immunology and Dagger  Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, § Pharmazentrum Frankfurt, Clinic of Goethe University, 60590 Frankfurt, Germany, and ** Department of Immunology, University of Maastricht, 6221 ER Maastricht, The Netherlands

Received for publication, January 15, 2002, and in revised form, April 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the capacity of mouse glomerular mesangial cells (MC) to express and function through two different low affinity Fcgamma Rs, the activating Fcgamma RIII and the inhibitory Fcgamma RII. Immunohistochemistry identified Fcgamma RII as the prominent Fcgamma R in the kidney, and low levels of Fcgamma RIIb2-specific mRNA were also detected in primary cultures of growth-arrested MC. Activation by tumor necrosis factor-alpha /interleukin-1beta induced substantial Fcgamma RII expression in proliferating MC. Importantly, however, stimulation with interferon-gamma (IFN-gamma )/lipopolysaccharide or IFN-gamma alone resulted in a complete down-regulation of Fcgamma RII, which was accompanied by a strong increase in FcRgamma chain mRNA and a surface appearance of Fcgamma RIII. Activating Fcgamma RIII triggered mRNA synthesis for monocyte chemoattractant protein-1 (MCP-1), MCP-5, cytokine-induced neutrophil chemoattractant, and RANTES, whereas Fcgamma RIII-deficient MC failed to respond to immune complex (IC) activation as shown by impaired production of MCP-1 mRNA/protein. In a passive model of acute anti-glomerular basement membrane (GBM) nephritis, induction of Fcgamma RIII and suppression of Fcgamma RII occurred in kidney tissues. Blockade of Fcgamma RII, when induced selectively in the kidney, resulted in enhanced inflammation. Taken together, our results define a novel regulatory pathway with opposite regulation of Fcgamma RII (suppressed) and Fcgamma RIII (induced) by IFN-gamma on MCs in vitro and anti-GBM IgG in vivo. Herein is provided the first evidence that glomerular Fcgamma RII plays an important immunoregulatory role in the initiation of IC glomerulonephritis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is compelling evidence for a primary pathogenic role of immune complexes (ICs)1 in many immunological diseases, such as systemic lupus erythematosus, rheumatoid arthritis, Goodpasture's syndrome, vasculitis, and glomerulonephritis (1-4). Although it had been assumed for a long time that IC diseases are mediated mainly by complement activation, the mechanisms by which IC formation triggers inflammation are still not fully understood. Spontaneous and induced mouse models of IC-dependent glomerular injury reveal an involvement of various complement components, including C1q, C3, and C5a (5-7). However, studies in FcRgamma chain-deficient mice define another potential pathway responsible for kidney damage in autoimmune glomerulonephritis requiring activating Fc receptors for IgG, Fcgamma Rs (8). The demonstration that both Fcgamma Rs and complement are essential in the experimental Arthus reaction suggests that the initiation of glomerulonephritis may also depend on the combined action of these two prominent effector systems (9-11).

Three classes of Fcgamma R are known on murine leukocytes: the high affinity receptor, Fcgamma RI, and the low affinity receptors, Fcgamma RII and Fcgamma RIII (12). Activating Fcgamma RI and Fcgamma RIII are expressed in association with the FcRgamma chain essential for triggering their various effector functions including antibody-dependent cell-mediated cytotoxicity, phagocytosis, and the production of proinflammatory molecules (13-15). In contrast, the two major isoforms of Fcgamma RII, Fcgamma RIIb1 and Fcgamma RIIb2, apparently lack such activating capacity but can inhibit FcRgamma -dependent activation signals when co-expressed on the same effector cell (16). This balance between activating/inhibitory Fcgamma Rs has been established in various murine models and is highlighted by an increased susceptibility of Fcgamma RII (-/-) mice to the pathogenic effects of antibodies in IC-triggered inflammation and autoimmune disease (17-20).

Glomerular mesangial cells (MC) are bone marrow-independent mesenchymal cells related to vascular smooth muscle cells (21, 22). Their contractility controls the rate of glomerular filtration and they are implicated in inflammatory and pathogenic processes of glomerular injury. When activated, MC are able to produce a variety of mediators, including nitric oxide, oxygen radicals, chemotactic cytokines, and several growth factors (23-25). This is accompanied by the induction of specific receptors, which enable them to form autocrine proliferative loops (26). Human MC, which are normally Fcgamma R-negative, can also be stimulated to express the activating receptors, Fcgamma RI and Fcgamma RIII. This induction of functional Fcgamma Rs may provide an important mechanism of initiation and progression of chronic glomerular inflammation by immune complexes (27, 28).

In the present study, we examined the capacity of mouse glomerular MC to express individual Fcgamma Rs by using primary cultures of MC established from normal and FcR-deficient mice. Moreover, we analyzed the role of the cytokines IFN-gamma , TNFalpha , and IL-1beta in the induction and/or down-regulation of the low affinity receptors Fcgamma RII and Fcgamma RIII on MC and in the IC/Fcgamma RIII-dependent generation of various proinflammatory mediators known to be involved in the pathogenesis of glomerulonephritis. By using this strategy, we first identified the b2 isoform of Fcgamma RII to be expressed on resting MC and normal kidney tissue. Second, we showed a requirement of IFN-gamma in changing the expression profile from inhibitory Fcgamma RII to activating Fcgamma RIII. Third, the induction of activating Fcgamma RIII on proliferating MC and the subsequent interaction with ICs triggered the enhanced synthesis of chemoattractants, including CC and CXC subfamily chemokines RANTES, MCP-1, MCP-5, and KC, respectively. Given the established function of these chemokines in renal IC inflammation (29-31), alterations in the levels of locally expressed inhibitory and activating Fcgamma Rs might be of pathogenic importance in glomerular disease in mice. This was verified in an experimental model of acute anti-GBM nephritis in which early renal mediator production and recruitment of neutrophils correlated with suppression of Fcgamma RII and induction of glomerular Fcgamma RIII. Moreover, the finding of increased MCP-1, MIP-2, and KC levels and augmented neutrophil influx upon local Fcgamma RII-blockade suggested that the function of the inhibitory Fcgamma RII and its down-regulation on mesangial cells during inflammation is critical in kidney pathology.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mice

The generation of Fcgamma RII (-/-), Fcgamma RIII (-/-) and FcRgamma (-/-) mice derived from embryonic stem cells with 129 origin and back-crossed for 8-12 generations with C57BL/6 mice has been described previously (13, 14, 16). Mice double deficient for Fcgamma RII and FcRgamma chain (Fcgamma RII/FcRgamma -/-) were purchased from Taconic (Germantown, NY). C57BL/6 mice were obtained from Charles River Laboratories (Sulzfeld, Germany). All these mice were used at 14-18 weeks of age for immunohistochemical analysis of kidney tissues (see below). All animal experiments were conducted in accordance with the regulations of the local authorities.

Immunohistochemistry of Mouse Kidney Tissue

To analyze the expression of individual Fcgamma Rs within the glomerulus, kidney tissue of Fcgamma R-deficient and C57BL/6 mice treated or not with increasing doses (50-500 ng) of intraperitoneal Escherichia coli LPS (Sigma) were processed for histological examination by immunocytochemical techniques as described (18, 32). Fcgamma R-positive staining was determined by incubating cryostat sections for 30 min with the anti-Fcgamma RII/III mAb 2.4G2 followed by incubation with the bridging antibody (Z0494) and the rat-APAAP antibody complex (D0488) for 30 min. The last two steps were repeated for 15 min followed by visualization using a Fast Red detection kit (Dako). All slides were counterstained with hematoxylin and mounted in Glycergel (Dako).

Mouse Glomerular Mesangial Cell Preparation and Characterization

Preparation-- Glomerular mesangial cells from C57BL/6 wild-type, Fcgamma RIII-deficient, and FcRgamma -deficient mice were prepared, characterized, and cultured according to the protocol described (23, 33). For each of the three independent preparations, 12 mouse kidneys were pooled. The kidneys were dissected from 6-week-old mice; the capsule and medulla were removed and the cortex cut into slices and rinsed. The material was sequentially pushed through 125- and 180-µm stainless steel sieves. The glomeruli separate from tubuli and Bowman capsules were recovered in a sieve with a 60-µm mesh screen and washed. By this procedure we obtained microscopically 95% pure, decapsulated glomeruli free of tubular contamination. These steps were followed by digestion with collagenase (1 mg/ml, type CLS4, 184 units/mg (Worthington)) for 30 min at 37 °C with shaking of the mixture every 5 min. After two washing cycles the digested glomeruli were seeded in 25-cm2 plastic culture flasks (Nunc) in a low volume (2 ml) of culture medium (see below) with 20% FCS to permit adherence. These conditions resulted in a predominantly mesangial outgrowth starting at day 6-12. Cells not resembling the typical growth in interwoven bundles were scraped off and removed together with the glomerular debris by gentle washing with fresh medium. Cells were then grown to confluence and switched to 10% FCS culture medium between the second and third passage.

Characterization-- Morphologically uniform cultures of stellate mesangial cells were used for characterization between the second and 12th passage. Light microscopic evaluation showed one layer of this cell type, with cell-cell contacts at discreet cell regions in a three-dimensional fashion resembling a net-like structure. Intercellular junctions were starting points for intracellular fibers, which by indirect immunofluorescence stained positively for myosin (see below). In mouse MC cultures extracellular matrix and "hillocks" (23, 25, 33) were visible by light microscopy. There was no morphological evidence of the presence of macrophages, dendrite-, endothelial-, or epithelial-like cells, or fibroblasts. However, to confirm the presence of exclusively intrinsic mouse mesangial cells of mesodermal origin we performed a series of biochemical, immune cytochemical, and functional tests. Briefly, these cells were resistant to puromycin treatment (10 µg/ml, 24 h), sensitive to mitomycin (10 µg/ml, 24 h). They stained positive by an indirect immunofluorescence technique for smooth muscle myosin, desmin, fibronectin, vimentin, and mouse MHC I antigen; and at confluence, they stained positive for extracellular deposits of collagen type IV and negative for factor VIII and cytokeratin. After the second passage, both dendritic cells and macrophages, which may represent 3-5% of glomerular cells in vivo, were carefully excluded as a contaminating subpopulation in MC cultures. Repeated MHC II antigen determinations with nonstimulated MC adherent on slides (with and without fixation) or flow cytometry (FACS) of suspended MC with anti-mouse I-Ad antibodies up to passage 12 were negative. Additionally, we did not detect any interferon activity in the MC supernatant, which might have been due to dendritic cells.

Culture Conditions and Stimulation for Mouse MC and Control Cells

Mouse MC used in passages 6-12 were cultured in RPMI 1640 medium supplemented with non-essential amino acids (1%), L-glutamine (2 mM), sodium pyruvate (2 mM), bovine insulin (5 µg/ml), beta -mercaptoethanol (45 µM), and endotoxin-free FCS (10%). Serum-free culture of mouse MC was performed using MCDB-302 medium (Sigma) with the supplements mentioned above, but without FCS, leading to growth arrest of MC after 48-72 h. Mouse macrophage J774 cells were used as control cells and cultured under standard conditions. All cell types were free of any mycoplasma contamination as tested every second week by a highly sensitive PCR analysis using previously established protocols (27). For the stimulating conditions for mouse MC, we used incubations for 48 h with the following concentrations of the various stimuli: 500-1000 units/ml mIFN-gamma (Roche Molecular Biochemicals), 500 units/ml mTNFalpha (Phillips, Bissendorf, Germany), 10 ng/ml recombinant human IL-1beta (Hoffmann-La Roche), 10 ng/ml mIL-10 (TEBU GmbH, Offenbach, Germany), 10 µg/ml E. coli LPS (Sigma).

RNA Isolation, RT-PCR Conditions, and Southern Blot Analysis

Total cellular RNA was isolated from mouse MC and J774 cells with RNA-Clean (AGS, Heidelberg, Germany) followed by LiCl2 precipitation and finally suspended in diethyl pyrocarbonate-treated water at a concentration of 1 µg/µl. Single-stranded cDNA was synthesized from 1.5 µg of RNA using reverse transcriptase and amplified by a modified Hot Start PCR protocol (27, 28) for a submaximal number of cycles (40 cycles for Fcgamma RI, Fcgamma RII, Fcgamma RIII, FcRgamma chain, MCP-5, and KC and 28 cycles for beta -tubulin) in a Varius VR thermocycler (Landgraf, Hannover, Germany). The primer sets used in PCR are given in Table I. PCR products were analyzed on 1.5% agarose gels. In all experiments, gels were blotted onto nylon membranes for Southern blot hybridization with 32P-labeled cDNA probes (Fcgamma RI, 714-bp fragment covering EC2 to TM/C exons (34); Fcgamma RII, 905-bp fragment covering TM to C3 exons of Fcgamma RIIb1 (35); Fcgamma RIII, 612-bp fragment covering the TM/C/UT exon (35); FcRgamma , KC, and MCP-5, all using their complete cDNA sequences (36-38)) to confirm specificity.

mAbs and FACS Analysis

The following FcR-recognizing antibodies were used: anti-Fcgamma RII/III (clone 2.4G2, rat anti-mouse IgG) (BD PharMingen, Heidelberg, Germany) and anti-Ly17.2 (clone K9.361, mouse anti-mouse IgG; kindly provided by Dr. U. Hämmerling, Sloan-Kettering Institute, New York), the latter specifically binding the Fcgamma RII antigen (18). Isotype control mAbs with irrelevant specificities were obtained from Immunotech (Hamburg, Germany). Binding of the mAb to the respective antigens was performed by indirect immunofluorescence. After mild trypsinization, mouse MC (106 cells) were incubated with the first mAb, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or goat anti-rat IgG F(ab')2 fragments (Dianova, Hamburg, Germany), and were analyzed on a FACScan flow cytometer (BD PharMingen).

Detection of Monomeric IgG2a Binding to Fcgamma RI on the Surface of Mouse MC

Cells were seeded on glass cover slides (8-well; Nunc, Roskilde, Denmark) and grown to subconfluency by covering the slides with 10% FCS medium in 10-cm Petri dishes. After stimulation with 500 units/ml IFN-gamma and 10 µg/ml LPS for 48 h, cells were washed twice with PBS, 0.05% Tween, 0.1% bovine serum albumin, fixed for 10 min at -20 °C with acetone/methanol (1:2), followed by three further washes with Tris-buffered saline. Cells were then incubated with monomeric mouse IgG2a at 37 °C for 30 min and phenotyped by the alkaline phosphatase anti-alkaline phosphatase technique (Dako).

Activation of Mouse MC by IgG Immune Complexes

In functional experiments, mouse MC induced to express or not the activating Fcgamma RIII were further stimulated with 50 µg/ml heat-aggregated IgG according to the methods of Santiago et al. (39) and Hora et al. (40). After 12 h of IgG IC activation, RNA was prepared and analyzed for chemokine mRNA synthesis by RNase protection assay. The culture supernatants were examined for the production of MCP-1.

RNase Protection Assay (RPA)

Assays for chemokine mRNA were conducted with the mCK-5 multiprobe template set according to the manufacturer's protocol (RiboQuant assay kit, BD PharMingen). The assay kit allows for the simultaneous detection of mRNA for each of the following mouse chemokines: lymphotactin, RANTES, eotaxin, MIP-1beta , MIP-1alpha , MIP-2, IFN-gamma -inducible protein-10 (IP-10), MCP-1, TCA3, and mRNA for the L32 and GAPDH housekeeping genes.

ELISA for MCP-1 Quantitation

Immuno-MaxiSorb 96-well microtiter plates (Nunc, Wiesbaden, Germany) were coated with 100 µl containing 1 µg/ml goat anti-mouse MCP-1 antibody (R&D Systems, Wiesbaden, Germany) in 100 mM sodium carbonate-bicarbonate buffer (pH 9.6) at 4 °C. After removing the excess capture Ab, the wells were filled with 200 µl of 1% bovine serum albumin in PBS and incubated at room temperature for 2 h to saturate excess binding sites. After three washes with PBS, serial dilutions of the experimental samples diluted in 1% bovine serum albumin/PBS were added to the plates and incubated at 4 °C overnight. After three washes, 100 µl of biotinylated detector anti-MCP-1 Ab was added to the wells and incubated for 4 h at room temperature. After three additional washes, the plates were incubated with peroxidase-conjugated streptavidin for 2 h. After three final washes, plates were developed with hydrogen peroxide and tetramethylbenzidine (Sigma) and stopped by the addition of 1 M H2SO4. Titrations of recombinant mouse MCP-1 (R&D Systems) were included in each experiment for standardization.

Experimental Anti-GBM Nephritis

Rabbit anti-mouse glomerular basement membrane (GBM) IgG antiserum was kindly provided by P. Heeringa. After decomplementation by heating at 56 °C for 30 min, 300 µl of anti-GBM antiserum was injected intravenously via the tail vein of ether-anesthetized mice. This concentration was effective to induce significant glomerular neutrophil influx in the early heterologous phase (2-4 h) of acute anti-GBM nephritis (58). In some experiments, glomerular Fcgamma RII was selectively blocked (30 min before anti-GBM treatment) by intracapsular injection of the neutralizing anti-Fcgamma RII Ly17.2 mAb (40 µg in 20 µl of sterile PBS) in one of the two kidneys per mouse. Mice were killed at 2 h after initiation of anti-GBM nephritis, and kidneys were assayed for glomerular expression of Fcgamma Rs, MCP-1 mRNA/protein production, MIP-2/KC CXC chemokine release, and neutrophil accumulation. The levels of Fcgamma R and MCP-1 mRNA in kidneys normalized to tubulin were quantitated by TaqMan real-time PCR using the following Fcgamma RII-, Fcgamma RIII-, MCP-1-, and beta -tubulin-specific primers and probes (Fcgamma RII: sense, 5'-AGTCTCCCTTGGCATTGGG-3', antisense, 5'-AGCATCCCTTGGACCAGGA-3', probe, 6-FAM-AAAGCAAGCCAGAAAGGCCAGGATCTAGT-TAMRA; Fcgamma RIII: sense, 5'-TGCAGCTCTTCCGAAGGCT-3', antisense, 5'-TGTCTTCCTTGAGCACCTGGA-3', probe, 6-FAM-TGGTGAAACTGGACCCCCCATGG-TAMRA; MCP-1: sense, 5'-CCAACTCTCACTGAAGCCAGC-3', antisense, 5'-CAGGCCCAGAAGCATGACA-3', probe, 6-FAM-CTCTCTTCCTCCACCACCATGCAGGT-TAMRA; beta -tubulin: sense, 5'-CACCATGAGCGGCGTCA-3', antisense, 5'-TTCGAAGGTCAGCATTAAGCTG-3', probe, 6-FAM-ACCTGCCTCCGTTTCCCGGG-TAMRA). Myeloperoxidase (MPO) serves as marker of neutrophil infiltration into kidney tissue and was assayed as described previously (9). In brief, homogenized tissue was suspended in 50 mM potassium phosphate buffer, pH 6, 0.5% hexadecyltrimethylammonium bromide, subsequently exposed to three freeze-thaw cycles, and finally sonicated. A total of 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide was added to the supernatant. The change in OD at lambda  = 450 nm was recorded. A serial dilution of MPO from human polymorphonuclear leukocytes (Calbiochem) served as a standard. Samples were run in duplicate. The concentrations of MCP-1, MIP-2, and KC in supernatants of homogenized kidneys were assayed in duplicate with MCP-1, MIP-2, and KC-specific ELISA kits (R&D Systems) according to the manufacturer's instructions.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constitutive Expression of the Inhibitory Fcgamma RII in Mouse Kidney-- Fcgamma R expression in the glomerulus was first determined by immunohistochemistry of kidney tissue sections obtained from FcR-deficient and C57BL/6 control mice. Weak anti-Fcgamma RII/III 2.4G2 mAb staining detectable in the kidneys of B6 mice was not observed in FcRgamma /Fcgamma RII (-/-) mice lacking surface expression of all known Fcgamma receptors, e.g. Fcgamma RI, Fcgamma RII, and Fcgamma RIII (Fig. 1A). The comparison of Fcgamma RIII (-/-) and Fcgamma RII (-/-) mice identified low constitutive expression of Fcgamma RII but not Fcgamma RIII in normal kidney (Fig. 1A). In Fcgamma RII (-/-) mice, anti-Fcgamma RII/III 2.4G2 mAb stainings became evident after intraperitoneal injection of 500 ng of LPS (Fig. 1B), indicating de novo renal expression of Fcgamma RIII. This glomerular appearance of Fcgamma RIII may be based on either infiltrating Fcgamma RIII-positive cells and/or induction of Fcgamma RIII on normally Fcgamma RIII-negative resident kidney cells.


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  		Fig. 1.   Different expression of low affinity Fcgamma receptors (Fcgamma RII and Fcgamma RIII) in kidney as assessed by immunohistochemistry. Kidney tissue sections from the indicated mice were stained with the anti-Fcgamma RII/III-specific 2.4G2 mAb and visualized using a Fast Red detection kit. A, staining intensity was similar in C57BL/6 and Fcgamma RIII (-/-) mice but was absent in Fcgamma RII (-/-) and Fcgamma RII/FcRgamma double-deficient mice (original magnification ×400). B, renal cortical tissue of Fcgamma RII (-/-) mice stained positive for 2.4G2 after intraperitoneal injection of 500 ng LPS (original magnification ×1000).

Fcgamma RII Gene Transcription in Resting Mouse MC under Basal and Stimulated Conditions-- We previously described a protocol in the generation of resting human MC (27, 41). This model system was therefore adopted to similarly induce growth arrest of mouse MC for up to 7 days in serum-free MCDB-302 medium without showing any irreversible phenotypic changes. Resting mouse MC were then incubated with or without IFN-gamma , IL-1beta , TNFalpha , TNFalpha /IL-1beta , IL-10, and LPS for 48 h. To quantitate mRNA expression of Fcgamma receptors, we performed RT-PCR with various Fcgamma Rs-specific primer pairs (Table I) and subsequent Southern blot hybridization. RT-PCR was controlled by beta -tubulin, which is not influenced by activation of MC, and RNA from J774 cells served as a positive control. Under basal conditions, resting mouse MC did not express any mRNA for activating Fcgamma RI and Fcgamma RIII (data not shown). In contrast, RT-PCR products of the expected size, specific for Fcgamma RIIb2 transcripts, could be amplified. Comparison with both the beta -tubulin control on MC and Fcgamma RII-specific mRNA in J774 cells indicated, however, that the abundance of Fcgamma RIIb2 was low (Fig. 2). From the stimuli mentioned above, the combination of IL-1beta and TNFalpha up-regulated Fcgamma RII gene transcription, whereas IFN-gamma and IL-10 led to a complete down-regulation (Fig. 2). When given alone, LPS, IL-1beta , and TNFalpha had only minor effects (Fig. 2, and data not shown). These results suggest that MC express basal levels of Fcgamma RIIb2 mRNA under nonproliferating conditions with no mRNA expression for activating Fcgamma Rs.

                              
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Table I
Primer sets used
Primers were selected by searching the published sequences of Fcgamma RI (34), Fcgamma RII (35), Fcgamma RIII (35), FcRgamma chain (36), KC (37), and MCP-5 (38).


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  		Fig. 2.   Fcgamma RIIb2 mRNA expression is adversely regulated by cytokines in resting MC. After 48 h of serum-free culture, growth-arrested MCs were either kept in serum-free medium or treated with the indicated stimuli for an additional 48 h. Per sample, 1.5 µg of total cellular RNA was subjected to RT-PCR and Southern blot hybridization. RNA from J774 cells served as a positive control. Ratios between the Fcgamma RIIb2 and beta -tubulin hybridization signals were determined using a Phosphoimager (Fuji) (the value obtained from the medium sample was set to 1 relative unit).

Proliferating Mouse MC Express Fcgamma RII Protein, Which Is Differently Regulated by IFN-gamma and IL-1beta /TNFalpha -- We recently showed by FACS analysis that antibodies detecting the mouse Ly-17.1/2 alloantigen system are specific for Fcgamma RII with no cross-reactivity to Fcgamma RIII (18). Now, we used the Fcgamma RII(Ly17.2)-specific mAb, K9.361 (42), to examine Fcgamma RII surface expression of mouse MC kept under proliferating conditions and stimulated cells with either IFN-gamma or a combination of TNFalpha and IL-1beta . As shown in Fig. 3A, proliferating unstimulated MC revealed surface Fcgamma RII protein at a low density that was substantially up-regulated by IL-1beta /TNFalpha . In contrast, MC became Fcgamma RII-negative after exposure to IFN-gamma for 48 h (Fig. 3B). These data were consistent with the RT-PCR analysis (Fig. 2) and demonstrated that the expression of inhibitory Fcgamma RII on glomerular mesangial cells is differently regulated by different cytokines.


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  		Fig. 3.   Surface expression of Fcgamma RII on mouse MC is differently regulated by TNFalpha /IL1-beta (A) and IFN-gamma (B). Proliferating cells were cultured for 48 h under 10% FCS medium conditions with or without the indicated stimuli. Surface expression of Fcgamma RII was analyzed by staining with anti-Fcgamma RII (Ly17.2)-specific mouse IgG mAb and FITC-conjugated goat anti-mouse F(ab')2. The fluorescence intensity of 10,000 cells was determined using a FACScan. The unfilled lines represent a negative control mAb.

IFN-gamma -dependent Down-regulation of Inhibitory Fcgamma RII Is Accompanied by Induced Expression of Activating Fcgamma Rs on Cycling Mouse MC-- IFN-gamma is capable of inducing or increasing activating Fcgamma RI and Fcgamma RIII receptors on proliferating human MC (27, 28). In mouse MC, low amounts of Fcgamma RI, Fcgamma RIII, and FcRgamma chain mRNAs were detected upon proliferation (Fig. 4). Both IFN-gamma and LPS, when given alone, increased mRNA expression (data not shown). The maximal stimulation with a combination of IFN-gamma /LPS (resulting again in abrogated Fcgamma RIIb2 mRNA) markedly activated FcRgamma chain transcription, suggesting a coupling between IFN-gamma -dependent Fcgamma RII down-regulation and induced expression of FcRgamma chain-associated Fcgamma Rs, Fcgamma RI and Fcgamma RIII, on mouse MC (Fig. 4). IFN-gamma -dependent induction of Fcgamma RIII protein was demonstrated by FACS analysis with the anti-Fcgamma RII/III 2.4G2 mAb (Fig. 5). The appearance of Fcgamma RI was examined more indirectly due to the lack of anti-Fcgamma RI-specific mAbs. Immunohistochemical analysis showed abundant staining of FITC-labeled monomeric IgG2a on IFN-gamma stimulated mouse MC with no staining on either unstimulated or TNFalpha /IL-1beta -treated MC (Fig. 6). Because Fcgamma RI is well established as interacting efficiently with monomeric IgG2a (12, 34), these findings may indicate that IFN-gamma induces, in addition to Fcgamma RIII, surface membrane expression of Fcgamma RI.


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  		Fig. 4.   IFN-gamma /LPS-dependent down-regulation of Fcgamma RII is accompanied by the strong induction of mRNA for the FcRgamma chain in proliferating MC. After 48 h of culture in 10% FCS-containing medium, proliferating MCs were treated or not with the indicated stimuli for an additional 48 h. Per sample, 1.5 µg of total cellular RNA was subjected to RT-PCR and subsequent Southern blot hybridization using beta -tubulin- and Fcgamma R-specific primer sets and cDNAs (see Table I) and analyzed using a Phosphoimager (Fuji). RNA from J774 cells was used as a positive control.


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  		Fig. 5.   IFN-gamma induced Fcgamma RIII surface expression on proliferating MC. Cells were cultured for 48 h under 10% FCS medium conditions with or without 500 units/ml IFN-gamma . Surface expression of Fcgamma RIII was analyzed by staining with the anti-Fcgamma RII/III-specific 2.4G2 rat IgG and FITC-conjugated goat anti-rat F(ab')2. The fluorescence intensity of 10,000 cells was determined using a FACScan. The unfilled lines represent a negative control mAb.


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  		Fig. 6.   IFN-gamma /LPS induced binding of monomeric IgG2a to proliferating MC. Mouse MC were grown to subconfluency on glass cover slides in 10% FCS culture medium for 48 h, and cells were stimulated or not with IFN-gamma /LPS or TNFalpha /IL-1beta for an additional 48 h. After fixation, MC were incubated with monomeric IgG2a, and binding was visualized by the alkaline phosphatase anti-alkaline phosphatase technique using Fast Red as the substrate for alkaline phosphatase. Positive and negative controls produced the expected results (original magnification ×400).

IgG Immune Complex Activation of Mouse MC-- To investigate the functional relevance of Fcgamma R expression in the activation of mouse MC, the effects of IgG ICs on the production of chemokines were measured by RPA, RT-PCR analysis, and ELISA. After induction of activating Fcgamma Rs by IFN-gamma or IFN-gamma /LPS (48 h), proliferating MC were subjected for 12 h to 50 µg/ml heat-aggregated IgG ICs. IFN-gamma itself or in combination with LPS induced a substantial accumulation of mRNA for MCP-1 and IP-10 but not lymphotactin, eotaxin, MIP-1beta , or MIP-1alpha in RPA (Fig. 7A, and data not shown), for MCP-5 and KC in RT-PCR (Fig. 7B) but only slightly for RANTES in RPA (Fig. 7A). The interaction of activating Fcgamma Rs with ICs led to the specific induction or increased synthesis of RANTES or MCP-1, MCP-5, and KC transcripts, respectively (Fig. 7). Moreover, MCP-1 protein levels of about 60 ng/ml were detected in the supernatants of MC cultures from control but not FcRgamma chain (-/-) mice (Fig. 7C), suggesting that increased expression of FcRgamma chain is required for IC-triggered MCP-1 release through activating Fcgamma Rs, Fcgamma RIII and/or Fcgamma RI. In support, IgG IC activation of MC that had been stimulated by TNFalpha /IL-1beta to express mainly Fcgamma RII did not result in such an enhanced chemokine production throughout all of the experiments (Fig. 7, A and C).


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  		Fig. 7.   Fcgamma RI/III-dependent chemokine production by MC. Proliferating MC from C57BL/6 or FcRgamma (-/-) mice were first incubated with the indicated stimuli for 48 h to either increase inhibitory Fcgamma RII (by TNFalpha , TNFalpha /IL-1beta ) or induce activating Fcgamma RI/III (by IFN-gamma , IFN-gamma /LPS) on the cell surface. After activation with heat-aggregated IgG IC, MC were analyzed for production of MCP-1, IP-10, and RANTES chemokine mRNAs by RPA (A), KC and MCP-5 mRNAs by RT-PCR (B), and MCP-1 protein by ELISA (C) (for details see "Experimental Procedures").

IgG IC-triggered Synthesis of MCP-1 Is Impaired in Fcgamma RIII-deficient MC-- To determine the role of Fcgamma RIII in the production of MCP-1 observed in mouse MC activated by ICs after IFN-gamma stimulation, we subjected MC from Fcgamma RIII (-/-) mice to IFN-gamma , and subsequently to IgG IC activation, and examined mRNA and protein levels of MCP-1. As shown in Fig. 8, Fcgamma RIII-deficient MC failed to respond to ICs with no induced synthesis of MCP-1. Impaired MCP-1 accumulation was similar to that seen in FcRgamma chain-deficient mice lacking both Fcgamma RI and Fcgamma RIII (Fig. 7C). Together, these data suggest that Fcgamma RIII is essential for IC-mediated MCP-1 synthesis by mouse MC in vitro with only a minor additional role for Fcgamma RI.


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  		Fig. 8.   IC-mediated synthesis of MCP-1 is impaired in Fcgamma RIII-deficient MC. Proliferating MC from C57BL/6 and Fcgamma RIII (-/-) mice were stimulated with 500 units/ml of either TNFalpha or IFN-gamma for 48 h and then activated with IgG IC for an additional 12 h. MCP-1 synthesis was examined at both RNA and protein levels by RPA (A) and ELISA (B). The results of RPA are expressed as the ratio between MCP-1 and GAPDH mRNA determined by densitometry using a Phosphoimager (Fuji).

Inverse Regulation of Glomerular Fcgamma RII and Fcgamma RIII in Acute Anti-GBM Nephritis-- Examination of the inflammatory processes in Fcgamma R (-/-) mice had previously shown that the activating Fcgamma RIII is an essential effector for neutrophil influx in the acute phase of anti-GBM nephritis (47). Because Fcgamma RII but not Fcgamma RIII receptors are normally expressed in MC and kidney tissue (Figs. 1-3), we now tested for modulation of renal Fcgamma RII and Fcgamma RIII expression in the initiation of experimental nephritis. Hereby, anti-GBM glomerulonephritis was induced by intravenous injection of 300 µl of anti-GBM IgG antiserum, leading to the formation of immobilized GBM:anti-GBM ICs in the glomerulus (58) followed by local MCP-1 production and early neutrophil influx that reached maximal levels of 223.9 ± 68.7 of µg MPO in kidney (n = 8, p < 0.001) at 2 h after challenge (Fig. 9, A and B). As assessed by TaqMan real-time PCR of kidney tissues, the Fcgamma RII/tubulin mRNA ratio strongly decreased from high levels of 147.9 ± 24.5 down to 74.0 ± 13.0 AU (arbitrary units) (n = 4; p < 0.05) with simultaneously enhanced amounts of Fcgamma RIII mRNA from low levels of 10.4 ± 2.9 up to 29.1 ± 0.8 AU (n = 4; p < 0.001) after the anti-GBM inflammatory insult (Fig. 9C). This regulatory principle of Fcgamma RII suppression and induced synthesis of Fcgamma RIII mRNA was equally seen in control experiments after intracapsular injections of 500 ng of LPS (Fig. 9D).


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  		Fig. 9.   Inverse regulation of glomerular Fcgamma RII/Fcgamma RIII in acute anti-GBM nephritis. IC nephritis was induced by intravenous application of 300 µl of anti-GBM IgG antiserum (+ anti-GBM) (A-C). Mice receiving 300 µl of sterile PBS served as negative controls (- anti-GBM). Mice receiving 500 ng of LPS served as positive controls (+ LPS) (D). After 2 h, kidneys were processed and assayed for neutrophil infiltration, as evaluated by a colorimetric MPO assay of kidney homogenate (A) for renal MCP-1 mRNA/protein production by TaqMan PCR and MCP-1-specific ELISA (B) and for glomerular expression of Fcgamma RII and Fcgamma RIII mRNA (also assessed by TaqMan PCR) (C and D). Data are expressed as mean ± S.E. obtained from 4-8 mice. Significance is determined by Student's t test (*, p < 0.05; **, p < 0.001).

Local Blockade of Glomerular Fcgamma RII Results in Enhanced Anti-GBM Inflammation-- The regulatory importance of the inhibitory Fcgamma RII had been established in mouse models of anti-GBM nephritis and Goodpasture's syndrome (20, 59). However, the issue of Fcgamma RII inhibition mediated by either infiltrating or local cells remained to be investigated. We thus designed in vivo experiments in which the inhibitory Fcgamma RII was blocked in a single mouse kidney by intracapsular injection of 20 µl (2 µg/µl) of purified anti-Fcgamma RII (Ly17.2) mAb 30 min before the induction of the anti-GBM nephritis. The analysis of inflammatory signs in anti-GBM-treated mice (n = 4) revealed augmented responses of neutrophil infiltration and chemokine production of MCP-1, MIP-2, and KC in the left kidney (receiving the anti-Fcgamma RII mAb) as compared with the right kidney (not receiving anti-Fcgamma RII mAb) of the same animal (Fig. 10). As a control, anti-GBM treated mice (n = 4) receiving an irrelevant isotype-matched antibody instead of the anti-Fcgamma RII mAb did not display such differences.


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  		Fig. 10.   Increased anti-GBM nephritis in kidney selectively treated with blocking anti-Fcgamma RII antibody. 40 µg of anti-Fcgamma RII mAb (black bars) or isotype control mAb (gray bars) was applied in the right kidney of mice followed by intravenous injections of anti-GBM IgG. After 2 h, MPO activity (A), MCP-1 (B), and MIP-2/KC chemokine levels (C) were determined in both kidneys of anti-GBM-treated mice by differentiating between the nontreated left kidney (left) and the mAb-treated right kidney (right). Shown are the mean values ± S.E. obtained from four mice each. Significance is determined by Student's t test (*, p < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies in Fcgamma R-deficient mice suggested the importance of Fcgamma Rs in promoting IC-mediated inflammation occurring at different tissue sites, including skin, lung, peritoneum, and kidney (12, 43-48). For example, FcRgamma -deficient mice had an attenuation of severe nephritis in models of spontaneous lupus nephritis and induced nephrotoxic serum glomerulonephritis (8, 46). Moreover, the activating Fcgamma RIII was found to be critical for glomerular neutrophil accumulation in acute GBM-anti-GBM IC nephritis (47). In contrast to the attenuated phenotype in Fcgamma RIII-deficient mice, the genetic deletion of the inhibitory Fcgamma RII augmented anti-GBM nephritis (20). These results are very similar to that obtained in other models of IC inflammation (17, 18), indicating that Fcgamma RIII-mediated activation responses are regulated by the inhibitory Fcgamma RII when co-expressed on the same effector cells. In this report, we now tested the concept that resident kidney cells express and function via activating and inhibitory Fcgamma Rs, thus providing a possible regulatory pathway for renal infiltration in IC-mediated nephritis. Immunohistochemical analysis of murine renal tissue revealed weak Fcgamma RII/III expression in glomeruli, attributed mainly to the non-activating Fcgamma RII in normal kidney and to Fcgamma RIII after challenge with LPS (Fig. 1). The source of induced Fcgamma RIII might be either activated resident kidney cells or, as recently suggested (47), infiltrating effector cells. Human mesangial cells can be induced to express functional Fcgamma RIIIA after stimulation with IFN-gamma and LPS (27). In preliminary experiments, Fcgamma R-deficient mice made chimeric by bone marrow transplantation pointed toward a substantial, although not exclusive, contribution of locally expressed Fcgamma Rs in the initiation of nephritis (data not shown). Thus, it is very likely that inhibitory/activating Fcgamma RII/III receptors on both infiltrating cells (neutrophils and monocytes) and intrinsic glomerular MC are important for mediating the glomerular response to IgG immune complexes.

An interesting observation is that the basal constitutive expression of Fcgamma RII observed in normal kidney could similarly be detected in primary cultures of MC (Figs. 2 and 3). RT-PCR revealed mRNA specific for Fcgamma RIIb2 but not Fcgamma RIIb1, which suggests that MC are related to macrophages, previously established to express Fcgamma RIIb2, which is able to mediate endocytosis of IgG immune complexes (35). In contrast to macrophages (49), however, the expression of Fcgamma RIIb2 on MC is tightly regulated. The combination of TNFalpha and IL-1beta increased Fcgamma RIIb2 on MC under both resting and proliferating culture conditions in vitro, whereas IFN-gamma induced complete down-regulation of Fcgamma RIIb2 mRNA and protein. Importantly, the inhibitory effect of IFN-gamma on Fcgamma RIIb2 expression inversely correlated with a strong accumulation of mRNA for the FcRgamma chain and increased mRNA levels of the alpha  chains for activating Fcgamma RI and Fcgamma RIII (Fig. 4). The pattern of induced activatory Fcgamma R gene expression appears to be specifically dependent on IFN-gamma , because it was not observed with TNFalpha and IL-1beta . Gene promoters of Fcgamma RI, Fcgamma RIII, and the FcRgamma chain contain IFN-gamma -responsive DNA elements (50-52), which further suggests that the IFN-gamma -dependent induction of Fcgamma RI/III expression is mediated by enhancement of gene transcription. The critical requirement of IFN-gamma for the appearance of Fcgamma RIII on the surface membrane of mouse MC was documented by FACS analysis (Fig. 5). We also observed strongly enhanced binding of monomeric IgG2a after stimulation with IFN-gamma /LPS (Fig. 6), which may indicate an induced surface expression of Fcgamma RI. However, because of the lack of specific anti-mouse Fcgamma RI antibodies, more direct proofs were not possible. This is in contrast to the situation for human MC, where IFN-gamma -dependent induction of functional Fcgamma RI (CD64) has clearly been demonstrated by the use of the CD64 mAb 22 (28, 53).

Chemokines play an important role in selectively recruiting certain subsets of leukocytes to specific sites of inflammation and tissue injury. Given the oppositely regulated expression of inhibitory Fcgamma RII versus activating Fcgamma RIII (and possibly Fcgamma RI) by IFN-gamma but not TNFalpha /IL-1beta , the effect of IgG IC activation for chemokine induction by MC was examined for the different cytokine combinations (Figs. 7 and 8). IFN-gamma -induced Fcgamma RI/III activated mRNA synthesis for CC and CXC chemokines, including MCP-1, MCP-5, RANTES, and KC. In contrast to IFN-gamma , the combination of TNFalpha /IL-1beta leading to increased expression of the inhibitory Fcgamma RII on MC resulted in only minor responses to IC activation. These results are similar to recent findings in other models, which demonstrated that Fcgamma RIII is an activating receptor, whereas Fcgamma RII is not (54). The contribution of activating Fcgamma RI in relation to Fcgamma RIII for enhanced chemokine production appears rather low, because the profoundly impaired synthesis of MCP-1 observed in MC from FcRgamma -deficient mice (lacking the expression of both Fcgamma RI and Fcgamma RIII) is equally seen in MC from Fcgamma RIII-deficient mice. Whether Fcgamma RI is indeed redundant awaits further investigation using the previously established Fcgamma RI-deficient mice (55). Taken together, our present in vitro results demonstrate that IFN-gamma is a potent stimulus for changing the expression profile from inhibitory Fcgamma RII to activatory Fcgamma RIII on MC, thereby enabling them to respond to IgG immune complexes via induced synthesis of MCP-1 and other chemokines which, as recently shown in several in vivo disease models (29, 31, 56, 57), are important mediators for renal infiltration.

In experimentally induced anti-GBM nephritis, it is striking to see that the ratio of glomerular Fcgamma RII and Fcgamma RIII expression is modulated during IC inflammation. The acute response triggered by GBM:anti-GBM IC formation leads to the production of MCP-1, MIP-2, and KC and subsequent neutrophil accumulation in the kidney and is associated with the down-regulation of renal Fcgamma RII and induced Fcgamma RIII expression (Fig. 9). Local intracapsular injections with LPS induce the same pattern of inverse Fcgamma R regulation. Considering the fact that the activating Fcgamma RIII is essential for neutrophil influx in anti-GBM-nephritis (47), the anti-GBM-dependent effect of inverse modulation of glomerular Fcgamma Rs may represent one of the initial events in the acute phase of this model of IC-induced glomerulonephritis. The genetic deletion of the inhibitory Fcgamma RII in Fcgamma RII (-/-) mice results in augmented inflammation in experimental models of anti-GBM nephritis and Goodpasture's syndrome (20, 59). However, it remained to be investigated whether Fcgamma RII-dependent inhibition is mediated by resident versus infiltrating effector cells. Our data demonstrate that functional and selective blockade of Fcgamma RII in kidney tissue is efficient in increasing neutrophil recruitment via enhanced production of CC/CXC chemokines (MCP-1, MIP-2, and KC) in acute anti-GBM nephritis in normal mice (Fig. 10). These findings strongly suggest that IC-dependent activation responses mediated by anti-GBM-induced Fcgamma RIII are normally controlled by the inhibitory Fcgamma RII expressed on kidney cells in vivo.

In summary, we have used primary cultures of glomerular mesangial cells from normal and FcR-deficient mice as an in vitro model of renal inflammation to investigate the relationship between cytokines and Fcgamma receptors in the regulation of IC-mediated chemokine production. This approach enabled us to demonstrate for the first time that the non-activating b2 isoform of Fcgamma RII is constitutively expressed in kidney and MC, thus providing a potential interaction with IgG immune complexes in the glomerulus. Similar to reports obtained from other species (27, 39), activating Fcgamma RI and Fcgamma RIII are normally not expressed under non-inflammatory conditions in mice. Interestingly, our observations establish the importance of murine IFN-gamma as an activator to promote IgG IC-mediated CC and CXC chemokine synthesis through the down-regulation of Fcgamma RII and induction of the activating Fcgamma RIII on MC. Furthermore, a major role of Fcgamma RIII over Fcgamma RI is suggested by a comparison between FcRgamma (-/-) and Fcgamma RIII (-/-) mice in the activation of the CC chemokine, MCP-1. Earlier reports have defined a contribution of MCP-1 and Fcgamma RIII in mouse models of nephrotoxic nephritis (47, 56), which may indicate that Fcgamma RIII and MCP-1 are critical components for the initiation of kidney disease in vivo. Our study adds to this point by identifying Fcgamma RIII-activated MC as an important cellular source of MCP-1 in vitro. Locally induced chemokine synthesis by cytokine-dependent expression of inhibitory/activating Fcgamma Rs in MC may thus be relevant for the regulation of cellular infiltration during nephritis. In support of this concept, initiation of acute anti-GBM nephritis is associated with induction of Fcgamma RIII and down-regulation of glomerular Fcgamma RII. Strikingly, the functional blockade of Fcgamma RII, when induced selectively in kidney, results in enhanced chemokine production and neutrophil influx. These findings establish that opposite regulation of and function through activating and inhibitory Fcgamma Rs on resident kidney cells, like mesangial cells, contribute to glomerular disease involving immune complexes in mice.

    FOOTNOTES

* This work was supported in part by Grants Ge892/5-1 and Ge892/7-2 from the Deutsche Forschungsgemeinschaft (to J. E. G.) and by Medizinische Hochschule Hannover Graduate Program funding (for E. N. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| These authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed: Abteilung für Klinische Immunologie, Medizinische Hochschule Hannover, CarlNeuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-3621; Fax:49-511-532-5648; E-mail: Gessner.Johannes@MH-Hannover.de.

Published, JBC Papers in Press, April 30, 2002, DOI 10.1074/jbc.M200419200

    ABBREVIATIONS

The abbreviations used are: IC, immune complex; Fcgamma R, IgG Fc receptor; GBM, glomerular basement membrane; IP-10, IFN-gamma inducible protein 10; KC, cytokine-induced neutrophil chemoattractant; MC, mesangial cell; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; RPA, RNase protection assay; RANTES, regulated on activation normal T cell expressed; IFN-gamma , interferon-gamma ; TNFalpha , tumor necrosis factor alpha ; IL, interleukin; mAb, monoclonal antibody(s); FCS, fetal calf serum; FACS, fluorescence-activated cell sorter; m, mouse; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MPO, myeloperoxidase; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription PCR.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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