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Summary The Fc receptor I for IgA (FcαRI) down-regulates humoral immune responses and modulates the risk of autoimmunity. This study aimed to investigate whether FcαRI targeting can affect progression of pristine-induced lupus nephritis. In the first experiment (early intervention), four groups of animals were evaluated: untreated FcαRI/FcRγ transgenic (Tg) mice and Tg mice administered control antibody (Ctr Fab), saline and anti-FcαRI Fab [macrophage inflammatory protein (MIP)-8a], respectively, three times a week for 29 weeks, after being injected once intraperitoneally with 0·5 ml pristane. In the second experiment, antibody injection started after the onset of nephritis and was carried out for 2 months, with similar groups as described above. MIP-8a improved proteinuria, decreased the amounts of glomerular injury markers, serum interleukin (IL)-6, IL-1 and monocyte chemoattractant protein (MCP)-1, and F4/80 macrophages in the interstitium and glomeruli, in both experiments. When MIP-8a was used as early intervention, a decrease in mouse serum anti-nuclear antibody (ANA) titres and reduced deposition of immunoglobulins in glomeruli were observed. This effect was associated with reduced serum titres of immunoglobulin (Ig)G2a but not IgG1, IgG2b and IgG3. Furthermore, pathological analysis showed lower glomerular activity index and less fibronectin in MIP-8a treated mice. This study suggests that FcαRI targeting could halt disease progression and lupus activation by selective inhibition of cytokine production, leucocyte recruitment and renal inflammation. Our findings provide a basis for the use of FcαRI as a molecular target for the treatment of lupus.

Summary Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An ‘efficiency’ index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P

Summary Myeloid FcαRI, a receptor for immunoglobulin (Ig)A, mediates cell activation or inhibition depending on the type of ligand interaction, which can be either multivalent or monovalent. Anti-inflammatory signalling is triggered by monomeric targeting using anti-FcαRI Fab or IgA ligand binding, which inhibits immune and non-immune-mediated renal inflammation. The participation of Toll-like receptors (TLRs) in kidney pathology in experimental models and various forms of human glomerular nephritis has been discussed. However, little is known about negative regulation of innate-immune activation. In the present study, we generated new transgenic mice that express FcαRIR209L/FcRγ chimeric protein and showed that the monovalent targeting of FcαRI exhibited inhibitory effects in an in vivo model of TLR-9 signalling-accelerated nephritis. Mouse monoclonal anti-FcαRI MIP8a Fab improved urinary protein levels and reduced the number of macrophages and immunoglobulin deposition in the glomeruli. Monovalent targeting using MIP8a Fab attenuates the TLR-9 signalling pathway and is associated with phosphorylation of extracellular signal-related protein kinases [extracellular signal-regulated kinase (ERK), P38, c-Jun N-terminal kinase (JNK)] and the activation of nuclear factor (NF)-κB. The inhibitory mechanism involves recruitment of tyrosine phosphatase Src homology 2 domain-containing phosphatase-1 (SHP-1) to FcαRI. Furthermore, cell transfer studies with macrophages pretreated with MIP8a Fab showed that blockade of FcαRI signalling in macrophages prevents the development of TLR-9 signalling-accelerated nephritis. These results suggest a role of anti-FcαRI Fab as a negative regulator in controlling the magnitude of the innate immune response and a new type of anti-inflammatory drug for treatment of kidney disease.

SummaryReceptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist – namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fcγ receptor family), IgE (FcεRI and CD23), IgA (CD89; Fcα/µR) and IgM (Fcα/µR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.

SUMMARYHuman red cells (RBC) coated with IgG anti-D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcγ R). Polymorphisms of Fcγ RIIa and Fcγ RIIIa affect IgG binding in vitro, and may alter the efficiency of clearance of immune complexes in vivo. In a RBC clearance study, 22 Rh D-negative subjects were given 100–400 µg human monoclonal or polyclonal IgG anti-D i.m. followed 48 h later by 51Cr-labelled D+ RBC. The half lives of the infused D+ RBC were determined, together with the coating levels of anti-D on the D+ RBC. Fcγ RIIA and FcγRIIIA genotyping was performed. Large ranges of phagocytosis and extracellular lysis of RBC in vitro, and of half lives of RBC in vivo, were observed. Clearance of RBC coated with monoclonal IgG3 anti-D (BRAD-3) was more rapid in five subjects homozygous for Fcγ RIIIa-F/F158 than in three subjects expressing the Fcγ RIIIa-V158 allele (P = 0·024). This effect was not observed, however, for those individuals given polyclonal anti-D. There was also no significant difference in the efficiency of RBC destruction in vitro or of RBC clearance in vivo between the subjects analysed for individual genotypes or alleles or combinations of alleles. In conclusion, the presence of the Fcγ RIIIa-V158 allele compromised the efficiency of removal of RBC coated with IgG3 anti-D.

SummaryCLB T3/4.A is a non FcR-binding CD3 mAb of the murine IgA isotype, which may be used as an alternative for the mitogenic OKT3 mAb in the treatment of acute cellular rejection after organ transplantation. We studied TCR signalling and T cell activation in response to T3/4.A in normal human PBMC in vitro. T3/4.A induced a rapid rise in free cytoplasmic Ca2+, not different from the response to mitogenic CD3 mAb. However, protein tyrosine phosphorylation and, particularly, MAPK activation, were reduced as compared to mitogenic CD3 mAb. T3/4.A enhanced expression of both CD69 and CD25, but proliferation and detectable cytokine production did not occur. Addition of either CD28 mAb or IL-2 induced a strong proliferative response, which was accompanied by cytokine production. At higher mAb concentrations, T cell activation decreased, which correlated with TCR downmodulation. To exclude the possibility that activation by T3/4.A depends on interaction of murine IgA Fc with as yet unknown FcR, we showed that also with CD3 mAb F(ab′)2 fragments upregulation of activation molecules occurred, as well as proliferation in the presence of costimulation. We conclude that the non FcR-binding murine IgA mAb T3/4.A acts as a partial agonist and leads to proliferation and cytokine production only in the presence of appropriate costimuli. These findings may explain the mitigated cytokine release syndrome observed in vivo with some nonmitogenic CD3 mAbs.

SUMMARY We analysed the biochemical features of receptors for the Fc-region of IgA (FcαR, CD89) on blood monocytes and granulocytes of patients with IgA nephropathy (IgAN). FcαR on monocytes of IgAN were found to have a higher Mr (60-80kD) than those of control monocytes (50–75 kD) and granulocytes (55–75 kD) in both IgAN and controls as shown by immunoprecipitation analysis. Removal of N-linked carbohydrates from FcαR on monocytes of IgAN revealed a 32–36 kD protein core, the Mr of which was still higher than that of controls (28–32 kD). When FcaL transcripts were analysed by reverse-transcription-PCR, only one prominent band was visualized in PCR products from IgAN monocytes. Since the results thus far show that IgAN monocytes express FcαR protein and mRNA differently from granulocytes and control monocytes, PCR products were then cloned and sequenced. The predominant band in PCR products from IgAN monocytes was identical to that of the FcαR a.l transcript, and an additional 10 transcripts containing five novel transcripts were obtained from granulocytes and control monocytes. In three transcripts, we found an insertion sequence between the S2 and EC1 domains, suggesting the existence of a new exon. These results suggest a predominant usage of FcαR a.l among various transcripts of FcαR in IgAN monocytes.

SUMMARY Patients with systemic lupus erythematosus (SLE) frequently have anti-lymphocyte autoantibodies, some of which also bind to surfaces of neurons. Since anti-ribosomal P protein autoantibodies (anti-P) from SLE patients also bind to surfaces of neurons, we hypothesized that anti-P are anti-lymphocyte antibodies. A panel of human T lymphocytes was evaluated for anti-P binding by indirect immunofluorescence. Affinity-purified anti-ribosomal antibodies were used as a source of anti-P. These autoantibodies bound to the surfaces of all transformed T cell lines tested. This binding was not mediated by Fc receptors. It was inhibitable by ribosomes. Anti-P bound to circulating T lymphocytes from healthy adults and children. They also bound to thymocytes and cord blood T cells from normal neonates. Circulating T cells from SLE patients with anti-P bound less anti-P than cells from healthy controls. Two patients were studied on multiple occasions. The capacity of their T cells to bind anti-P correlated inversely with titres of anti-ribosomal antibodies. Anti-ribosomal antibodies, other than anti-P, also appear to bind to T cells. The surface of T cells contains a protein with the size and antigenicity of the ribosomal P protein, P0. We conclude that anti-ribosomal antibodies are a subset of anti-lymphocyte autoantibodies. Their possible role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE has to be defined in further studies.

SUMMARY IgA nephropathy (IgAN) is defined by the predominant deposition of IgA immune complexes (IC) in the glomerular mesangium. Interaction between IgA immune complexes and mesangial cells (MC) could be a linchpin for the genesis of IgAN. We studied the modulation of MC expression of IgA receptors (FcαR) by selected cytokines. Binding of 125I-IgA to quiescent human MC showed 2.55 ×105 sites/cell with an affinity (Ka) of 3.2 ×107 M−1. Addition of selected recombinant cytokines had no significant influence on Ka, but increased the number of sites/cell relative to unstimulated cells. Northern hybridization using the pHuFcαR cDNA probe showed time-dependent increases in mRNA expression in stimulated versus control cells. IL-6 and tumour necrosis factor-alpha (TNF-α) had a biphasic effect on the FcαR mRNA level; at 48 h, IL-6 increased steady state mRNA levels about six-fold relative to control, TNF-α increased mRNA four-fold, and interferon-gamma (IFN-γ) induced FcαR mRNA two-fold. By reverse transcriptase-polymerase chain reaction (RT-PCR), the FcαR expressed on human MC appears highly homologous to that expressed by U937 cells. Altered FcαR expression in response to cytokines may influence the pathogenesis of IgAN by affecting deposition and/or clearance of IgA-IC in the mesangium.

SUMMARY Human polymorphonuclear cells (PMN) were found to adhere to a novel model of blood vessel wall-associated IgG. The internal surfaces of cellulose acetate hollow fibres, of comparable internal diameter to small blood vessels, were coated with normal serum human IgG, heat-aggregated IgG (HAIgG), laminin or fibrinogen. Under conditions of flow mimicking those in a small vessel. PMN were found to adhere markedly only to immunoglobulin-coated fibres. Arrest on HAIgG was inhibited by excess soluble IgG but not by bovine serum albumin (BSA), demonstrating that the adhesion was IgG-specific and presumably mediated by FcγR on the PMN surface. Pre-adsorption of serum components onto HAIgG-coated fibres enhanced PMN arrest, due most probably to fixation of complement components by immobilized HAIgG, resulting in additional potential to entrap PMN via complement receptors such as CR3. Treatment of PMN with the regulatory neuropeptide substance P also enhanced adhesion to HAIgG-coated fibres and caused increased surface expression of FcγRI, FcγRII and FcγRIII. A mouse cell line derived from L cells, hR4C6, stably transfected with human FcγRII, was found to adhere under flow lo HAIgG-coated fibres, whilst untransfected parent L cells did not. This adhesion was similarly inhibited by excess soluble IgG, confirming the capability of FcγR to mediate cell arrest. The study strongly suggests that FcγR may play an important role in intravascular PMN arrest and we speculate that in inflammatory diseases PMN may adhere via FcγR to immobilized immunoglobulin on the vascular endothelium, with subsequent degranulation and tissue damage.

Organ transplantation offers the potential to improve quality of life and prolong survival for increasing numbers of patients with organ failure, and bone marrow or peripheral stem cell transplantation may constitute the only curative approach for patients with select haematological diseases. Since the early sixties, heterologous polyclonal sera against human thymocytes or lymphocytes (ATG or ALG) are employed for immunosuppression, but significant progress in transplantation medicine was achieved with the introduction of novel oral immunosuppressive agents like calcineurin inhibitors, mycophenolate mofetil and – more recently – sirolimus. However, acute rejection episodes or high risk clinical situations, e.g. in pretransplanted patients may require even more pronounced immunosuppression. In these situations, several monoclonal antibodies against defined antigens broaden the therapeutic armentarium today [1]. Among these are antibodies against the IL-2 receptor (CD25) or CD3, and antibodies against other antigens are expected to follow along this line [2].

SUMMARY The low affinity IgE receptor FcɛRII (CD23) is important in several aspects of T and B cell function. In this study serum levels of soluble CD23 (sCD23) were measured in three groups: 26 female patients with systemic lupus erythematosus (SLE), 21 females with primary Sjögren's syndrome (pSS) and 25 normal healthy females. The concentration of sCD23 was determined using an enhanced chemiluminescent sandwich ELISA developed in this laboratory. Increased levels of sCD23 were observed in pSS and in SLE patients compared with controls (median 23·0 versus 8·6, P

SUMMARY The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (FcγRI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgGI anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecfic antibody in passive immunotherapy.

SUMMARY The expression of a range of surface molecules/receptors that are important in the host response to infection and foreign antigens was examined using peritoneal macrophages isolated from patients on continuous ambulatory peritoneal dialysis (CAPD) with peritonitis. The macrophage phenolypic profile was compared with that of normal peripheral blood monocytes. Consistently there was increased expression by macrophages of CD14, ICAM-1 (CD54). FcγRI (CD64). FcγRII (CDw32), FcγRIII (CD 16), transferrin receptors (CD71)and tissue factor. Increased expression of MHC class II was marginally significant. There was no detectable expression of either the p55 (CD25) or p70 chains of the IL-2 receptor. The expression of the complement receptors. CRI (CD35) and CR3 (CD11b. CDI8), was reduced. The activity of well-known inflammatory cytokines, rather than uraemic molecules, can account for the phenotypic profile of these extravasated peritoneal macrophages. The results of this study indicate that peritoneal macrophages from CAPD patients with peritonitis display a phenotype consistent with them being in vivo-derived inflammatory macrophages, and that they arc appropriate for use in studies of anti-inflammatory agents.

SUMMARYThe European collaborative study of HIV-infected pregnant women in Europe now indicates a 13% risk of fetal HIV infection (originally thought to be about 30%, and possibly higher in some countries). Several reports suggest trans-placental passage. However, the detailed mechanisms associated with such vertical transmission have not yet been clarified. We have examined the possibility that HIV enters placental tissue from maternal blood via binding to CD4 and Fc receptors (FcR) at the trophoblast level, allowing intraplaccntal infection. Here we report the detection of several FcR with distinct localization in the placental villus as well as CD4 surface expression on human trophoblast cells. In addition, we show that trophoblastic cells interact specifically with the gp120/gp160 viral envelope protein. By their tissue localization, these receptors could be responsible for the entry of HIV into the fetal placental cells. Furthermore, purified placental cells can be directly infected by HIV in vitro, and the infection is inhibited by soluble CD4. This suggests a crucial role of the CD4 receptor but an additional way of entry cannot be excluded. Such an in vitro model may be suitable for further studies concerning placental HIV transmission and its prevention.

SUMMARYSoluble forms of low affinity Feγ receptors (FcyR), also called IgG-binding factors (IgG-BF), have been shown to play a regulatory role in immune responses. By using an immunodot assay with the anti-mouse FcyR MoAb, 2.4G2, the levels of IgG-BF have been measured in the sera of mice bearing syngeneic tumours of lymphoid or non-lymphoid origin or in mice injected with high doses of murine IgG. These sera contained large amounts of IgG-BF as compared with controls. In the case of mice bearing IgG2a- or IgG2b-secreting hybridomas or lymphomas, serum IgG-BF increased progressively with tumour size and serum monoclonal IgG concentration, reaching 4–12 times the normal levels. A less than three-fold increase was found in mice bearing an IgG1-secreting hybridoma or tumours which do not secrete IgG (IgA-secreting hybridoma, non-immunoglobulin-secreting lymphoid tumours or melanoma) or in mice injected with 9 mg of monoclonal IgG2a. The enhancement of serum IgG-BF levels was independent of the expression of FcγR by the tumour cells, suggesting that the majority of IgG-BF secreted in response to tumours was produced by the host rather than by the tumour. The increased production of IgG-BF may participate in the control of tumour growth and in the modulation of the host immune responses in tumour-bearing animals.

In vitro Fc epsilon RII expression was examined in patients with atopic dermatitis, those with non-atopic eczematous dermatitis and normal individuals following stimulation of peripheral blood cells with recombinant IL-4 (rIL-4), phytohaemagglutinin (PHA), or PHA plus rIL-2. At various days cells were stained with MoAbs to human lymphocyte Fc epsilon RII and to lymphoid cell-surface antigens and analysed by flow cytometry. rIL-4, but not rIL-2, specifically induced Fc epsilon RII on T cells as well as B cells in atopic dermatitis, eczematous dermatitis and normal individual groups. Both atopics and non-atopics generated comparable proportions of Fc epsilon RII+ T cells (T epsilon cells), whereas the frequency of B cells bearing Fc epsilon RII(B epsilon cells) was significantly higher in patients with extensive atopic dermatitis than in those with mild atopic dermatitis and other subjects. Comparable levels of T epsilon cells were detected in both atopic and non-atopic donors following stimulation of peripheral blood cells with PHA or pre-activation of the cells with PHA plus subsequent incubation with rIL-2. Whereas both CD8+ and CD4+ subsets were present in T epsilon cell populations induced specifically by rIL-4, PHA and PHA plus rIL-2, patients with atopic dermatitis had a greater tendency for Fc epsilon RII expression on CD8+ T cells compared with patients with eczematous dermatitis and normal individuals. Recombinant interferon-gamma (rIFN-gamma), but not rIFN-alpha or prostaglandin E2 (PGE2), suppressed the generation of T epsilon cells by rIL-4 in atopics and non-atopics to the same degree. These results suggest the aberrant control of Fc epsilon RII expression on T cells, especially those bearing CD8, in atopic dermatitis.

SUMMARY Skin biopsies of 31 non-atopic patients, 20 with mycosis fungoides, six with psoriasis and five with contact dermatitis, and of five non-atopic healthy controls were compared for the presence of cell-bound IgE and vacant IgE binding sites. IgE+ cells were demonstrated in the cutaneous infiltrate of nine (45%) patients with mycosis fungoides, two (33%) with psoriasis and one (20%) with contact dermatitis. Following pre-incubation of skin sections wilh IgE myeloma protein to saturate vacant IgE-binding sites, 14 out of 16 patients (88%) with stage I mycosis fungoides, five (83%) patients with psoriasis and one (20%) with contact dermatitis showed an increase in the number of IgE+ cells. While cell-bound IgE was positively related to serum IgE levels the expression of IgE-binding sites was not. All IgE+ cells were HLA-DR+ dendritic cells identified as either macrophages (CD68+, CDI4+) or Langerhans cells (CDl+). Skin biopsies of non-atopic healthy controls or clinically uninvolved skin in mycosis fungoides had neither any IgE+ cells nor any vacant binding sites. Inhibition studies with IgGl, IgG4 and IgE myeloma proteins as well as with several enzymatic fragments of igE demonstrated that IgE interacled with Fcɛ-receptors through isotype-specific structures on the Fcɛ-fragment. Four anti-CD23 monoclonal antibodies, however, were unuble to stain vacant Fcɛ-receptors nor could they block IgE-binding. We hypothesize that locally-secreted lymphokines, like IL-4 or interferon-γ, induce Fcɛ-receptors on dendritic cells in the cutaneous infiltrate and that these receptors become occupied in parallel wilh elevated serum IgE levels.

SUMMARYGrowth of a methylcholanthrene-induced fibrosarcoma in BALB/c mice was accompanied by an increase in the activation state of tumour-associated macrophages (TAM). as measured by their FclgG receptor expression, phagocytic index and β-glucuronidase levels. All of these parameters were markedly higher in TAM than in peritoneal macrophages (PM) derived from the same animal. On the other hand, PM from tumour-bearing mice showed lower activation parameters than PM from normal animals. We also studied the effect on tumour development of three inhibitors of prostaglandin synthesis: indomethacin. piroxicam and aspirin. Intraperitoneal administration of these drugs during 8 d was followed by the regression of palpable tumours. Indomethacin (90 mg/d) induced 45% regression, while with piroxicam (two 400 mg/d doses and six 200 mg/d doses) and aspirin (1 mg/d) 32% and 30% regressions, respectively, were observed. The growth rate of non-regressing tumours, which had reached different volumes by the end of the treatment, was delayed to a similar extent by the three anti-inflammatory non-steroidal drugs (NSAID). With respect to TAM, the treatment did not induce any significant change in their activation state, though both piroxicam and indomethacin increased slightly the TAM number. In contrast, NSAID administration was followed by a remarkable increase in the activation parameters of PM when compared with PM from tumour-bearing mice receiving no treatment. Indeed, these parameters were in some cases higher than those of PM from normal mice. The leukocytosis (60 000/μl) with neutrophilia (80%) induced by tumour growth on peripheral blood leukocytes (PBL) was reversed by the treatment to values close to normal, in parallel with the reduction of tumour size. A drop in haematocrit was also noted which was most probably a consequence of tumour growth rather than of the treatment. This study reveals that the three NSAID tested have a remarkable antitumour activity, which correlates with the restoration of PM activity and PBL values.

The characteristic finding of high levels of circulating immune complexes in patients with the autoimmune connective tissue diseases systemic lupus erythematosus (SLE) or scleroderma has raised the possibility that these patients may have a primary defect in immune complex clearance. The Fc receptor for IgG (Fc gamma R) plays a central role in the phagocytosis of antibody complexes. We have analysed Fc gamma R (type II) RFLPs identified in TaqI- and MspI-restricted genomic DNA and found that their distribution in SLE and scleroderma did not differ significantly from controls. Hybridization with specific regions of the Fc gamma RII cDNA clone indicate that part of the Fc gamma RII alpha locus is duplicated in some individuals. A further Fc gamma RII gene has recently been identified (Fc gamma RII alpha'). This gene shows greater than 95% homology with Fc gamma RII alpha and may thus be the candidate gene for the apparent alpha duplication seen in some individuals. It is possible that an individual may possess one, two, three or four TaqI Fc gamma RII alpha/alpha' alleles, correlating with incidence and numerical heterogeneity in Fc gamma RII alpha and alpha'. The physiological effects of this numerical heterogeneity remain to be investigated.

SUMMARY Marrow aspirates from 11 patients with megaloblastic haemopoiesis and from 14 healthy individuals with normoblustic haemopoiesis were studied for antibodies associated with polychromatic/orthochromatic erythroblasts, using an 125I-labelled anti-human immunoglobulin reagent and autoradiography. In addition, the expression on these cells of receptors for FcIgG (FcR) and of the type I receptor for fragments of the third complement component (CR1) were investigated with receptor-specific monoclonal antibodies. 125I-labelled anti-mouse immunoglobulin and autoradiography. The percentages of immunoglobulin-positive erythroblasts were significantly greater in the megaloblastic than in the normoblastic marrows. Abnormally high percentages of labelled erythroblasts were present in patients without any manifestations of an autoimmune disorder. The percentage of labelled erythroblasts in the marrows of the patients correlated well with the degree of anaemia. FcR were absent on the majority of megaloblasts or normoblasts while the expression of CRI was similar in both types of cell. The difference between the percentage labelling of megaloblasts and normoblasts was therefore unlikely to be due to greater binding of immune complexes with or without associated complement to mcgaloblasts than normoblasts. The megaloblast-hound immunoglobulin is, therefore, likely to have recognized abnormally expressed epitopes on the surface of megaloblasts. The results suggest that natural autoantibodies play a role in the destruction of erythroblasts during megaloblastic haemopoiesis.

SUMMARY It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10–12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10–12 days, major cell expansion had occurred and they were essentially a pure (>90%) CD3+CD16-CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (> 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (

SUMMARYPolyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG 2–10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgGl and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRIII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.

SUMMARY The proliferative response of T cells during autologous mixed lymphocyte reactions (AMLR) was affected by depletion of IgG Fc receptor+ T lymphocytes (Tg). Removal of Tg cells resulted in enhanced proliferation, and EA-rosette isolated Tg cells, when added to AM LR cultures as irradiated third components, reduced the uptake of 3H-thymidine by 63–87% in a dose-dependent manner. Negative selection using an avidin-biotin affinity chromatography technique demonstrated that the suppression was mediated by DR+ Tg cells; the major proportion of which also expressed the CD8 antigen. By comparing AMLR supcrnatants collected from control (lacking Tg) and suppressed (containing Tg) cultures on days 2, 3, and 4, it was established that supernatants from suppressed cultures had significantly reduced levels of cytokine activity. These data indicate that the CD8 +/DR+ Tg cells function as suppressor cells during an AMLR and reduce the proliferative response by inhibiting AMLR responder T cells from producing the cytokines necessary for in vitro growth.

SUMMARYActivation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-1 (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM-1, recognized by a monoclonal antibody (MoAb) ENAI, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab)2 fragments of MoAb ENA1 up to 60%. An additive inhibition of the adhesion was established by pre-incubation of the PMN with anti-CD18 MoAb and J. or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENAI, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD 18 MoAb and J. or LAI reduced the adhesion observed if intact ENAI were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD18 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a roie in graft rejection and in diseases where antibodies directed against endothelium are involved.

Monocytes and neutrophils share a common progenitor and perform many similar functions, as reflected in the expression of several shared membrane molecules. We show here that such molecules can be independently regulated by the cytokines interferon-gamma (IFN-gamma) and the colony-stimulating factors (CSF) in the context of the cell type on which they are present. Thus, IFN-gamma causes neutrophils to express the high-affinity receptor for IgG, Fc gamma RI, which has been considered to be a monocyte-specific receptor. Although neutrophil Fc gamma RI never reaches the levels present on monocytes, it is induced more rapidly and by lower amounts of IFN-gamma than monocyte Fc gamma RI. Of the CSF, only macrophage (M) CSF has an effect which is to cause a decrease in expression of monocyte Fc gamma RI. Secondly, after culture monocytes express Fc gamma RIII which is constitutively expressed by neutrophils. Although neutrophil Fc gamma RIII can be readily modulated by IFN-gamma, granulocyte (G) CSF and granulocyte-macrophage (GM) CSF, these cytokines do not alter the low levels of monocyte Fc gamma RIII. Thirdly, expression of the gp55 protein recognized by CD14 monoclonal antibodies is decreased after exposure of monocytes to IFN-gamma. Neutrophils express low levels of CD14 and, in this case, IFN-gamma causes an increase in the CD14 antigen on these cells. Of all the molecules investigated, only HLA class II is confined to monocytes, with increases in expression induced by IFN-gamma but not by the CSF.

SUMMARYThe cell surface expression of low-affinity Fc epsilon receptor (FceRII), which is identical to CD23, and the serum levels of IgE binding factors (IgE BFs), a soluble form of FceRII, and IgE were investigated in HTLV-I-infected individuals, and the results were compared with those for HTLV-I-negative controls. The occurrence of FcεRII+ mononuclear cells did not differ among the blood samples from the HTLV-I-infected groups; however, the levels of serum IgE BFs were significantly decreased (P

SUMMARYThe human IgA Fc receptor (FcαR, CD89) plays an important role in host defence against invading pathogens. To study the properties of the receptor, 12 MoAbs, namely, MIP7c, MIP8a, MIP9a, MIP10c, MIP11c, MIP14b, MIP15b, MIP38c, MIP59c, MIP65c, MIP68b and MIP71a, were generated. The inhibitory effects of the antibodies on FcαR functions were tested. Three of the antibodies, MIP7c, MIP8a and MIP59c, were able to block up to 90% of soluble FcαR binding to IgA-coated beads and 70–80% of neutrophil phagocytosis of IgA immune complexes (IC). MIP8a could also inhibit IgA IC-induced neutrophil lactoferrin release, while cross-linking of FcαR with MIP8a and anti-mouse IgG could elicit neutrophil lactoferrin release. However, IgA IC-induced lactoferrin release required both extracellular calcium and magnesium, whereas MIP8a-induced release did not require extracellular magnesium and only partially required extracellular calcium. In addition, the time course of IgA IC-induced lactoferrin release was slow. Lactoferrin was not detectable if the incubation time was less than 0·5 h. In contrast, MIP8a-induced lactoferrin release was fast. Lactoferrin could be detected within 5 min of incubation. Therefore, neutrophil lactoferrin release induced by IgA IC differed from that induced by cross-linking of FcαR with MIP8a.

SUMMARYIn order to analyse the specificity of human anti-ribosomal P protein antibodies, anti-ribosomal P protein antibodies were affinity-purified from the sera of lupus patients. Their binding capacity towards recombinant SmD protein and recombinant SmB/B′ protein was evaluated by immunoblot and ELISA. Epitope mapping of SmD was performed by means of synthetic peptides. Anti-ribosomal P protein antibodies bound recombinant SmD (5/10) and SmB/B′ (4/10) on immunoblot; 6/10 showed binding capacity to SmD on ELISA. Inhibition experiments using ELISA confirmed the specificity of this binding. Our data indicate the cross-reactivity of spontaneously developed anti-ribosomal P protein antibodies with the B/B′ and D constituents of the Sm complex. The coexistence of anti-Sm and anti-ribosomal antibodies in lupus sera may therefore be due, at least in part, to the reactivity of a single autoantibody population.

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL-1 and TNF-α accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL-1 receptor antagonist (IL-1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL-1 activity. The present study shows that human serum IgA induces significant IL-1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL-1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL-1β release, and even inhibited LPS-induced IL-1β release. Induction of IL-1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of FcαR with MoAb MY-43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA-induced IL-1Ra production, suggesting that FcαR-mediated induction of tyrosine phosphorylation is required for the IgA-induced stimulation of IL-1Ra release. In addition, triggering of FcαR with MoAb specifically down-regulated TNF-α and IL-6 release in human monocytes activated with Hib. By the induction of IL-1Ra and down-regulation of the release of inflammatory cytokines such as IL-1β, TNF-α and IL-6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.

SUMMARY Parameters of immune activation/differentiation were studied in a group of newly diagnosed HIV− and HIV+ pulmonary tuberculosis (TB) patients. Compared with controls, HLA-DR expression on both CD4 and CD8 T cells from the HIV− TB patients was approximately doubled; HLA-DR on T cells from the HIV+ group was tripled. The monocytes from both groups of patients expressed abnormally high levels of the Fcγ receptors I and III. Serum levels of tumour necrosis factor-alpha (TNF-α), neopterin and β2-microglobulin were increased in HIV− and even more so in HIV+ TB patients. The expression of HLA-DR on T cell subsets and of FcγR on monocytes correlated with each other, but not with serum activation markers. This pattern of non-specific activation during TB infection may be associated with enhanced susceptibility to HIV infection.

SUMMARY Data from a previous study suggested that peripheral blood monocytes in patients with rheumatoid arthritis (RA) may be activated. Therefore, in this study we sought further evidence of ‘presynovial’ activation of monocytes. Our results show that phenotypic changes are demonstrable in peripheral blood monocytes in patients with RA, including increased expression of CR3 (CDllb/CD18) and FcRI (CD64). However, changes are most extensive in synovial monocytes/macrophages and especially for HLA-DR and intercellular adhesion molecule-1 (ICAM-1) (CD54). We conclude that monocyte/macrophage activation is most evident within the joint, and that ‘presynovial’ changes occur but are of limited extent.

SUMMARYWe examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C′-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C′-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADF of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE: seems to be more efficient in enhancing HIV-I replication than C′-ADE. While FcR-ADE leads to increased internalization of HIV-1. C′-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE arc reactive with the gp120 viral envelope antigen, whereas antibodies involved in C′-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADH and C′-ADE may contribute lo the spread of HIV-1 from mother to the fetus.

A group of Venezuelan patients with SLE showed an increased proportion of responders to Leu-4, an anti-CD3 MoAb of the IgG1 class, compared with ethnically matched non-SLE patients and healthy controls. The rate of proliferative responses or IL-2 production induced by MoAb Leu-4, and the helper effect of macrophages from Leu-4 responders on T cells from a third-party donor were comparable in patients and controls. No significant differences in the binding of murine IgG1 molecules by macrophages from SLE patients and controls were observed. The proportion of monocytes/macrophages expressing Fc gamma RI was significantly higher in SLE patients. However, the expression of FcRII, the type capable of supporting Leu-4-mediated responses, and of Fc gamma RIII was comparable in monocytes from SLE patients and controls. Our results suggest that Venezuelan patients with SLE may have a genetic predisposition for the expression of the phenotypic variant of Fc gamma RII capable of binding murine IgG1 molecules.

SUMMARY Quantification of human peripheral blood NK subsets has been made in a group of Kenyan adults and children with acute P. falciparum malaria. Results were compared with data obtained from three age- and sex-matched control cohorts: parasitaemic but asymptomatic children: aparasitaemic children and adults; and adult Caucasians with no previous history of malaria. Separated NK subsets were tested in vitro for cytotoxicity to erythrocytic sehizonts of P. falciparum in the presence and absence of cytokines. There was a statistically significant quantitative and qualitative depression of the CD3- CD56+ subset in patients with acute malaria and this was accompanied by an expansion of the ‘non-functional’ CD3- CD57+ CD16- CD56- subset. Both CD3- CD16+ and CD3- CD56+ NK cells from all patients and donors lysed schizonts. and this cytotoxicity was enhanced by the addition of recombinant interferon-alpha and or IL-2, notably with the CD3- CD56+ subset. Interestingly, asymptomatic donors had the highest levels of CD3- CD56+ NK cells, which also demonstrated an enhanced response to cytokine stimulation. Cytotoxicity to schizonts was accompanied by the release of soluble NK cell lytic factors. Neomycin suppressed cytotoxicity in a dose-dependent manner, Indicating that the lysis of schizonts by NK cells involves phospholipase C-mcdiated phosphoinositide metabolism. Our findings define a role for NK cells in immunity to malaria through the lysis of infected erythrocytcs as a first-line defence against the parasite.

Cells expressing Fc receptors for IgE (Fc epsilon RII) were identified in the peripheral blood from patients with atopic dermatitis and with eczematous dermatitis, and normal non-atopic subjects by using monoclonal antibodies to human lymphocyte Fc epsilon RII, and to lymphoid cell-surface antigens by immunofluorescence staining. Based on the extent of the dermatitis patients were classified as severe (greater than 50% skin surface involved), moderate (50-10%) and mild (less than 10%). Patients with severe and moderate atopic dermatitis had 5.9% and 5.7% Fc epsilon RII+ peripheral blood mononuclear cells (PBMC), respectively, that were significantly higher than percentages in mild atopic dermatitis patients (2.6%), severe to moderate eczematous dermatitis patients (2.3%), mild eczematous dermatitis patients (2.2%) and normal individuals (1.7%)(0.05 greater than P). In severe and moderate atopic dermatitis patients, 10% of Fc epsilon RII+ PBMC were T cells that preferentially expressed CD8, and the remainder B cells and monocytes. Fc epsilon RII+ T cells comprised 1% of peripheral T cells, while half or more of peripheral B cells expressed Fc epsilon RII. In mild atopic dermatitis patients, eczematous dermatitis patients and normal subjects. Fc epsilon RII were expressed exclusively on 25-35% of peripheral B cells. Short-term treatment and long-term follow-up of atopic dermatitis patients revealed that changes in the skin condition were related closely to fluctuations in the proportion of Fc epsilon RII+ PBMC. Total serum IgE levels and atopic respiratory allergy did not influence the percentage of Fc epsilon RII+ PBMC. These findings suggest that the percentage of Fc epsilon RII+ PBMC reflects the extent of atopic dermatitis.

SUMMARY B lymphocytes from patients with systemic lupus erythematosus (SLE) secreted high levels of immunoglobulin spontaneously when cultured in vitro. Addition of the cytokines interleukin-2, interleukin-4 and interleukin-6 either alone or in combination failed to augment spontaneous immunoglobulin synthesis. Percoll-separated low-density SLE B lymphocytes matured into immunoglobulin-secreting cells also independent of exogenous interleukins. During maturation these cells became enlarged and less dense, and began to express CD23. This was in contrast to normal B cells, which did not secrete immunoglobulin spontaneously but synthesized IgM after interleukin stimulation. These results indicate that in vitro immunoglobulin synthesis by SLE B cells is already initiated in these cells and progresses independently of further stimulatory manoeuvres.

SUMMARYMonoclonal antibodies (MoAbs) that are able 10 discriminate between dendritic cells (MoAb RFD1+) and mature macrophages (MoAb RFD7+ (in normal tissues were used in combination with density separation techniques to isolate relatively homogeneous subpopulations of macrophages from human bronchoalveolar lavage (BAL). A characterization of surface antigen expression, and functional capacity was then carried out on each isolated alveolar macrophage (AM) subset. One population with the phenotype RFD1+ RFD7- obtained from the non-adherent cell pool showed the characteristics of antigen-presenting cells having absent or poor expression of Fc and C3b receptors, a low content of lysozomal hydrolase and poor phagocytic capacity. This population strongly stimulated T lymphocytes in allogeneic mixed lymphocyte reactions (MLR). A second AM population, isolated by adherence and density centrifugal ion expressed the phenotype RFD1+ RFD7+. These cells showed the same phenotypic characteristics of mature macrophages with strong expression of C3b and Fc receptors, and marked phagocytic capacity. Such AM were very poor stimulators of allogeneie MLR. Under certain circumstances the RFD1+ RFD7+ cells were shown to actively repress the stimulatory capacity of the RFD1+ RFD7- subpopulation. These results suggest that variations within the functional capacity of AM subsets may be capable of influencing the strength of acquired T cell immune responses of the lung.

SUMMARY Mucosal ulceration of the oral, and to a lesser extent genital tissues is an essential feature of Behçet's syndrome and is associated with changes in the IgA class of immune responses. Indeed, a significant increase in the proportion of cytophilic IgAl was found in circulating CD8 and CD4 cells (P < 0.01), with a corresponding decrease in IgA-Fc receptors on these T cells. Furthermore, 30–40% of the cytophilic IgA1 on T cells may have been of the polymeric secretory type and the rest of the monomeric variety. IgA isotype of B cells was also significantly increased (P < 0.001), without an overall change in circulating B cells. However, a surprising finding was the significant up-regulation of γŞ T cell receptor in the CD8 (P < 0.01) in the absence of a change in the proportion of αβ T cell receptor. The results suggest that some common microbial antigen might initiate at the mucosal surface an immune defence reaction characterized by T cells with γΔ receptors and IgA-specific B cells. However, IgAl bound to circulating T cells may down-regulate the central T cell function.

SUMMARYWe have confirmed our previous observation that improved human renal allograft survival is associated with the presence in pre-transplant serum of a high molecular weight lymphocyte Fey receptor-blocking factor. Serum fractionation studies suggest that this factor is a complex protein consisting of IgG together with an IgG-binding protein which has an apparent molecular weight of approximately 60 kD.

SUMMARY Autoimmunity may be due to augmentation of immune responses by human CD8 cells which bind the lectin Vicia villosa (VV). We have investigated T cells in rheumatoid arthritis (RA) by double immunofluorescence flow cytometry, in order to assess VV-binding CD8 and CD4 cells from the peripheral blood and synovial fluid. A significant increase in CD8+ VV adherent (P

The expression of class II MHC encoded antigens (HLA-DR) and Fc gamma receptors by peripheral blood monocytes from untreated patients with small cell carcinoma of the bronchus was compared with that of normal donors. Fc gamma receptor expression was found to be elevated in these patients in comparison with normal. In contrast HLA-DR antigen expression by patients' monocytes was somewhat depressed in comparison with normal. Continuous intravenous infusion of a total of 400 megaunits/m2 of human lymphoblastoid interferon-alpha (IFN-alpha) over a 5 day period markedly increased both monocyte HLA-DR antigen expression and Fc gamma receptor expression in comparison with that of untreated patients and normal donors. The initial increases declined somewhat but were still evident after 3 weeks of intermittent intramuscular IFN-alpha therapy.

Peripheral blood lymphocytes (PBL) from rheumatoid arthritis (RA) patients and healthy controls (NC) were assayed for Fc receptors by four methods. Three of the four assays showed RA PBL to contain a greater proportion and number of Fc receptor-bearing cells than PBL from healthy controls. This confirmed our earlier report and indicated that the proportion of Fc receptor-bearing cells detected can be influenced by the test system. Neither the proportion of B, nor the non-B, non-T lymphocytes was consistently increased in RA PBL, suggesting that the increase found results from an increase in Fc gamma receptor-positive T cells.

The high incidence of infections characteristic of impaired cell-mediated immunity in patients with hairy cell leukemia led us to study T lymphocyte function in sixteen patients with the lymphocyte transformation test. All patients showed imparied responses to mitogens, attributable to one or more of the following causes: dilution of responsive T cells by inert hairy cells, shortage of monocytes to give adequate interaction with the T cells and a significant decrease in the number of T cells with Fcmu receptors proportional to the percentage of hairy cells in the peripheral blood. The response to antigens was severely depressed; PPD was one of the few antigens that induced positive reactions in half the cases. We conclude that in patients with hairy cell leukaemia, T lymphocyte function, as tested in a proliferative assay, is severely impaired and that this may contribute to the deficient resistance to infection.

Using a low density culture technique which allows normal human peripheral blood T cells to proliferate actively in vitro when stimulated by mitogens, it is shown that dexamethasone enhances the spontaneous induction of cells bearing receptors for the Fc of IgM. Dexamethasone has no effect on the induction of cells bearing receptors for the Fc of IgG.

The Fc receptor mediated reaction, the beta-glucuronidase and the lactic dehydrogenase activities of monocytes and the serum lysozyme level were tested together with the circulating immune complex content of patients with systemic lupus erythematosus. Simultaneously with the increasing FC receptor-mediated reaction and the elevated enzyme activities of patient monocytes, the secretion of lysozyme and the immune complex content of the sera were higher than those of the controls. A positive correlation was demonstrated between the Fc receptor-mediated reaction, the beta-glucuronidase activity, the lysozyme secretion and the immune complex content of the sera. Thus, the monocytes of patients appeared to be activated by the circulating immune complexes.

Mononuclear cells were isolated from peripheral blood and analysed with T and B markers (E rosettes and SIg) and on contaminating monocytes and PMN. The suspensions contained 63.3 +/- 4.8% T lymphocytes, 11.4 +/- 3.8% B lymphocytes, 9.0 +/- 5.5% null lymphocytes, 15.1 +/- 3.8% monocytes and 1.1 +/- 0.6% PMN. Of all cells, 27.6 +/- 12.1% formed EA rosettes with OR1R2 red cells coated with anti-CD Ripley. In preparations fixed after cytocentrifugation, the EA rosette-forming cells were studied with regard to esterase activity. The proportion of cells with detectable Fc receptors was further studied in purified T lymphocyte and in monocyte suspensions. Finally, EA rosettes were isolated by gradient centrifugation and the rosette forming cells studied with conventional T and B markers. All procedures gave corresponding results: on average 11-14% of the T lymphocytes and nearly 100% of the null cells formed EA rosettes, while only 2% of the B lymphocytes had detectable Fc receptors. Of the purified monocyte and PMN populations, on average 72 +/- 10.5 and 14.5 +/- 5.4%, respectively, formed EA rosettes. Thus, the Ripley assay, representing an important marker for null lymphocytes, cannot be regarded as specific for this population of white blood cells.