The antiinflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. The modulation of macrophage migration and eicosanoid metabolism

Dietary polyunsaturated fatty acid modulation exerts a beneficial effect in immune-mediated glomerulonephritis. To elucidate the mechanisms underlying this phenomenon, the effects of essential fatty acid (EFA) deficiency on the heterologous phase of nephrotoxic nephritis in rats (induced by the injection of a rabbit antiglomerular basement membrane antibody) were studied. The heterologous phase of nephrotoxic nephritis was characterized by an invasion of leukocytes into the glomerulus. Polymorphonuclear neutrophils predominated early on (3 h), whereas macrophages predominated at 24 and 72 h. EFA deficiency selectively prevented the influx of macrophages into the glomerulus. The invasion of polymorphonuclear neutrophils, in contrast, was unaffected. The influx of leukocytes into the glomerulus during nephritis was accompanied by a marked enhancement (10- to 40-fold) in glomerular thromboxane and leukotriene B4 production. EFA deficiency largely attenuated this change. Renal dysfunction during the heterologous phase of nephritis was manifested as azotemia, polyuria, sodium retention, and proteinuria. With EFA deficiency, polyuria, azotemia, and sodium retention were not seen. Proteinuria was reduced by approximately 85%. To address whether the lack of macrophage migration into the glomerulus in the context of nephritis with EFA deficiency might be due to a functional defect in macrophage migration, the chemotactic responsiveness of EFA-deficient macrophages was examined. EFA-deficient macrophages displayed normal chemotactic migration toward activated C. In sum, EFA deficiency prevents the invasion of macrophages into the glomerulus in nephrotoxic nephritis and attenuates the accompanying metabolic and functional alterations, but does not affect macrophage chemotactic responsiveness. Alterations in macrophage elicitation and lipid mediator generation by inflamed glomeruli thus appear to be central to the salutary effect of dietary polyunsaturated fatty acid modification on glomerulonephritis.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Triggering of the superoxide generation of macrophages by crosslinking of Fc gamma receptor

Crosslinking of monomeric IgG2 molecules bound to the Fc gamma receptors on the cell surface of guinea pig macrophages generated the triggering signal for the superoxide-generating system. A binding experiment indicated that macrophages have saturable binding sites for monomeric IgG2. Scatchard analysis of the binding data showed that macrophages have an average of 4 X 10(5) binding sites per cell and the association constant for the binding was 4.2 X 10(6) M-1. Binding of monomeric IgG2 to macrophages could be detected by subsequent reaction with the 125I-labeled F(ab')2 fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG2 monomer to Fc receptor did not stimulate superoxide release, further addition of the F(ab')2 fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide, and the amount released was dependent on the dose of cell-bound IgG2. When macrophages were bound with a constant dose of IgG2 monomer in the first step, the superoxide release triggered by the addition of the F(ab')2 of anti-guinea pig Fab was dependent on the dose of the F(ab')2 fragment added. These results show that crosslinking of Fc receptors triggers the superoxide generation.     

Interferon gamma induces in human neutrophils and macrophages expression of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I or CD64)

Immune interferon (IFN-gamma) induces in human neutrophils accumulation of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I, CD64) with a mechanism that is independent from de novo protein synthesis and from activation of the Na+/H+ antiporter. Monocyte-derived macrophages can also be induced to express high levels of Fc gamma R-I mRNA by IFN-gamma, without requirement of protein synthesis. Unlike what is observed in neutrophils, induction by IFN-gamma of macrophage Fc gamma R-I mRNA was significantly depressed by the Na+/H+ antiporter inhibitor amiloride. These results indicate that phagocytes' Fc gamma R-I mRNA induction by IFN-gamma is regulated by different mechanisms depending on the target cells.     

Anti-glomerular basement membrane antibody-induced experimental glomerulonephritis: evidence for dose-dependent, direct antibody and complement-induced, cell-independent injury

The immunopathogenesis of anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) involves the triad of antibody fixation to the glomerular basement membrane, local complement activation and polymorphonuclear leucocyte (PMN) infiltration. We sought to investigate the potential for contributions to renal injury from each component of this triad (i.e., antibody, complement, and PMN). This study compares the ability of antibody to induce injury in normal, PMN-depleted and dual (i.e., PMN + complement)-depleted rabbits by assessing the quantity of kidney-fixed antibody (KFA) necessary to induce proteinuria. (The KFA levels are expressed in micrograms of antibody bound per gram renal cortex.) Normal animals developed significant proteinuria at KFA 60 +/- 6 micrograms/g. PMN-depleted animals were injury free at KFA 60 +/- 6 micrograms/g, but developed heavy proteinuria at KFA 140 +/- 5 micrograms/g. The protection from injury by PMN depletion at lower KFA levels confirms the important contribution of PMN to injury in this model. Furthermore, the demonstration of a clearcut threshold for injury despite PMN-depletion indicates that glomerular antibody deposition and complement activation can cause direct PMN-independent injury. Dual depletion studies showed a significantly higher KFA threshold for injury in PMN + complement-depleted animals than in PMN-depleted, complement intact animals (195 +/- 10 micrograms/g, cf. 140 +/- 5 micrograms/g; p less than 0.01). The occurrence of injury despite dual mediator depletion demonstrates that antibody itself can produce direct, complement and PMN independent renal injury. Together the PMN depletion and dual depletion studies indicate that antibody-induced glomerular complement activation produces direct, PMN-independent renal injury that can be prevented by complement depletion. Thus each of the potential mediators studied is individually capable of producing renal injury in anti-GBM-GN. In addition to the well established injurious role of PMN, this study demonstrates that glomerular complement activation and anti-GBM antibody deposition may both produce neutrophil-independent components of renal injury. The relative contributions of the individual mediators to injury is dependent on the quantity of antibody deposited within the glomerulus.     

Il-4 therapy prevents the development of proteinuria in active Heymann nephritis by inhibition of Tc1 cells

The role of IL-4, a key Th2 cytokine, in promoting or inhibiting active Heymann nephritis (HN) was examined. HN is induced by immunization with Fx1A in CFA, and proteinuria in HN is associated with subepithelial IgG and C3 deposition and infiltration of CD8(+) T-cytotoxic 1 (Tc1) cells and macrophages into glomeruli, as well as induction of Abs to Crry. Treatment with rIL-4 from the time of Fx1A/CFA immunization stimulated an earlier IgG1 response to Fx1A, induced anti-Crry Abs, and up-regulated IL-4 mRNA in lymphoid tissue, but did not alter proteinuria. Treatment with MRCOx-81, an IL-4-blocking mAb, resulted in greater proteinuria, which suggests endogenous IL-4 regulated the autoimmune response. Delay of rIL-4 treatment until 4 wk post-Fx1A/CFA immunization and just before the onset of proteinuria prevented the development of proteinuria and reduced Tc1 cell infiltrate in glomeruli. Delayed treatment with IL-4 had no effect on titer or isotype of Abs to Fx1A or on Ig, C3, and C9 accumulation in glomeruli. Treatment with rIL-13, a cytokine that alters macrophage function such as rIL-4, but has no direct effect on T or B cell function, reduced glomerular macrophage infiltrate, but did not prevent proteinuria or CD8+ T cell infiltrate. Anti-Crry Abs were paradoxically only induced with rIL-4 therapy, not in HN controls with proteinuria. It was concluded that the rIL-4 effect was probably by inhibition of Tc1 cells, which normally mediate the glomerular injury that results in proteinuria.     

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.     

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

TNF-alpha up-regulates renal MIF expression in rat crescentic glomerulonephritis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that participates in the pathogenesis of endotoxemia and experimental crescentic glomerulonephritis. However, very little is known about how MIF production is regulated in disease. We therefore examined whether tumor necrosis factor alpha (TNF-alpha), a known inducer of MIF expression by macrophages in vitro, up-regulates local and systemic MIF expression in a macrophage-mediated rat model of crescentic glomerulonephritis. MATERIALS AND METHODS: Anti-glomerular basement membrane (GBM) glomerulonephritis was induced in groups of six primed rats. Animals were treated with 1 mg/kg soluble TNF-alpha receptor (TNFbp) or saline from the time of disease induction until they were killed on Days 1, 7, or 14. Renal MIF expression was assessed by in situ hybridization, immunohistochemistry, and ELISA, and compared with macrophage accumulation and indices of renal damage. RESULTS: Although TNFbp treatment on Day 1 of the disease had only a partial effect upon the up-regulation of glomerular MIF expression, on Days 7 to 14 it almost completely abrogated the increase in glomerular and interstitial MIF mRNA and protein expression. In addition, TNFbp treatment significantly inhibited MIF secretion by cultured glomeruli and reduced serum MIF levels. The inhibition of renal MIF expression was paralleled by a significant inhibition of glomerular and interstitial macrophage infiltration (p < 0.001 versus saline treated), a significant suppression of renal injury (proteinuria and serum creatinine), and a marked reduction in histologic damage (glomerular hypercellularity, crescent formation, and interstitial fibrosis; all p < 0.01 versus saline treated). CONCLUSIONS: This study demonstrates for the first time that TNF-alpha up-regulates local MIF expression by both infiltrating macrophages and resident kidney cells in rat crescentic glomerulonephritis. In addition, TNF-alpha regulates systemic MIF production. Thus, TNF-alpha, together with MIF, may play a pathological role in immunologically induced renal disease.     

Role of novel rat-specific Fc receptor in macrophage activation associated with crescentic glomerulonephritis

Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.     

Immunoglobulin treatment suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion

Atherosclerosis is associated with immune activation. Immunoglobulin is used for the treatment of immune-mediated diseases. The mechanisms and importance of the Fc portion of immunoglobulin upon experimental atherosclerosis in apolipoprotein E-deficient mice were examined. Experimental atherosclerosis was induced in mice fed a high-fat diet containing 0.3% cholesterol. Over 8, 12, and 16 wk, on alternate days, mice were treated with an intraperitoneal injection of either 1 g.kg-1.day-1 of human intact immunoglobulin or F(ab')2 fragments of human immunoglobulin. Fatty streak formation and fibrofatty plaques were markedly suppressed in mice that received intact immunoglobulin for 8, 12, and 16 wk. In contrast, atherosclerotic lesions were not ameliorated in mice that received F(ab')2 fragments. Immunohistochemical analysis revealed that macrophage accumulation in the fatty streak lesions was suppressed in mice received intact immunoglobulin but not in those that received F(ab')2 fragments. In addition, the cytotoxic activities of splenocytes from immunoglobulin-treated mice, but not from F(ab')2 fragment-treated mice, were significantly suppressed compared with those from human serum albumin-treated mice. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. Immunoglobulin therapy markedly suppressed atherosclerosis due to Fc receptor-mediated anti-inflammatory and immunomodulating actions. The antiatherosclerotic effects of immunoglobulin may be related to the suppression of cytotoxic activity of atherogenic T cells and the reduction of macrophage accumulation in the lesions.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

Further evidence for the antibody nature of C3 nephritic factor (C3NeF).

C3 nephritic factor (C3NeF), found in the sera of some patients with membranoproliferative glomerulonephritis, has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. Purified C3NeF binds to the amplification convertase of complement, C3b,Bb, and thereby prevents decay of its C3-cleaving potential. The capability of C3NeF to bind to C3b,Bb was used as a means for purifying C3NeF to homogeneity. The investigation described in this report suggests that binding of C3NeF to C3b,Bb occurs via the Fab portion of the molecule. Pepsin treatment of eight C3NeF preparations resulted in an average loss of 76% of C3NeF functional activity. Papain treatment induced a loss of approximately 90%. The decrease in functional activity could be attributed to the accelerated rate of dissociation of 125I-F(ab')2 and 125I-Fab fragments from stabilized cell-bound C3b,Bb. The dissociation rate of 125I-F(ab')2 from C3b,Bb was comparable with the decay of the functional activity of C3b,Bb stabilized by F(ab')2 or Fab fragments of C3NeF. Although these results suggest that the stabilizing activity of C3NeF is mediated by the Fab portion of the molecule, it was found that the Fc portion also contributes to its functional activity.     

Regulation by Fc fragments of the secretion of collagenase, PGE2, and lysozyme by mouse peritoneal macrophages

Fc fragments of human IgG can stimulate resident mouse macrophages in culture to secret collagenase, to increase PGE2 secretion, and to decrease the secretion of lysozyme. Active synthesis and secretion were shown by the progressive accumulation of these products in the extracellular medium and inhibition of secretion by cycloheximide. A dose-dependent effect of Fc fragments was demonstrable. Brief exposure of cells to Fc fragments was sufficient to cause the macrophages to secrete collagenase and large amounts of PGE2 for prolonged periods of time, suggesting that a sustained activation rather than temporary modulation of the cells had occurred. Con A had similar effects on macrophage secretory activity. These findings indicate that proteins that bind to specific macrophage plasma membrane receptors may stimulate the secretion of products that promote the inflammatory response.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.