Flow cytometry with crystal violet to detect intracytoplasmic fluorescence in viable human lymphocytes. Demonstration of antibody entering living cells

A new method is described for the detection of intracytoplasmic fluorescence and its differentiation from surface staining of viable human lymphocytes using flow cytometry after addition of crystal violet which quenches surface but not internal fluorescence. This has then been used to study antibody penetration of viable human lymphocytes, using FITC-conjugated IgG from normal serum or serum containing anti-RNP antibody. The results showed that 54 +/- 1% normal lymphocytes were penetrated by anti-RNP antibody and 23 +/- 3% by normal IgG respectively. The lymphocyte population analysed by flow cytometry has been directly demonstrated to be viable by FDA staining. These results provide unequivocal evidence that antibody can penetrate viable human lymphocytes.     

Antibody penetration of viable human cells. II. Anti-RNP antibodies binding to RNP antigen expressed on cell surface, which may mediate the antibody internalization.

As U1 small nuclear ribonucleoprotein (U1 snRNP2) has a crucial role in pre-mRNP splicing, the interaction of anti-RNP antibody with snRNP within viable lymphocytes may profoundly influence cell functions. We have shown that antibody can penetrate viable human lymphocytes, and anti-RNP antibodies enter more cells than other anti-nuclear antibodies or control IgG. In order to study the in vitro interaction of anti-RNP antibodies with viable cells, T lymphocytes were metabolically labelled with 35S-methionine, then incubated with the antibodies and washed. A set of 35S-labelled cell-associated snRNP polypeptides A, B'/B, C and D were found to bind to both monospecific human polyclonal anti-RNP IgG (human anti-RNP IgG) and a mouse monoclonal anti-RNP antibody (2.73), indicating that anti-RNP antibodies interacted with RNP antigen inside or/and on the surface of viable cells. To investigate antibody binding to RNP antigen on the cell surface, the cell surface proteins were either iodinated with 125I or the cells processed for immunoelectron microscopic studies after incubation with MoAb. At least seven 125I-labelled polypeptides on the cell surface were found to be immunoprecipitated by the anti-RNP MoAb which have similar molecular weights to U snRNP polypeptides 70K, A, B, D, E, F, and G. The immunoelectron microscopic studies showed that the gold particles formed clustered patches on the cell membrane. Further studies suggested that RNP antigen bound to the cell surface, and the RNP binding structure was probably a heterodimer receptor. This study provides evidence to suggest that anti-RNP antibody entry into viable cells may be mediated by interaction with RNP antigen expressed on the cell surface.     

A major subpopulation of Fc receptor-bearing lymphocytes revealed by rosette formation in dextran media: studies with pig, sheep and rat lymphocytes

Blood lymphocytes from young pigs which formed 9.1 +/-0.7% Fc rosettes (mean +/- S.E., range 4.1-16.0), with rabbit and pig immunoglobulin-coated indicator cells used in optimum conditions in phosphate-buffered saline (PBS), formed 32.6 +/- 1.8% (16.9-44.3) in the presence of 4% dextran (DFc). The proportion of DFc+/Fc+ lymphocytes varied from 2.2 to 5.9 (3.8 +/- 0.3). Compared with the PBS test, in dextran rosettes are formed with more lymphocytes and with red cells coated with less immunoglobulin. Ficoll at 14% gave similar enhancement. Dextran enhancement of Fc rosettes was also observed with sheep PBL, but not with rat thoracic duct lymphocytes. The FC portion of IgG is responsible for both Fc and DFc rosettes. Thus most Fc and DFc rosettes, formed with serum antibody-coated bovine RBC, are revealed by IgG coated but not F(ab')2-coated BRBC, and show dose-dependent inhibition in the presence of free pig IgG (complete at 5-10 mg/nl) but not IgM (up to 2 mg/ml). Separation of lymphocytes on nylon wool suggests that DFc rosettes are formed by B cells and some T cells. Over half of the weak Fc rosette-forming lymphocytes (DFc-Fc) elute in the non-adherent fraction, which contains few B cells, and therefore are a subpopulation of T cells.     

Immunoglobulin Fc receptor molecules on Xenopus laevis splenocytes.

The existence of membrane-associated Fc receptors for IgY (Fc nu R) or IgM (Fc mu R) was demonstrated on a large percentage of Xenopus splenocytes. The Fc receptors were detected by direct fluorescent staining in which the spleen cells were incubated with fluorescein isothiocyanate (FITC)-conjugated antigen-complexed IgY antibodies or with FITC-conjugated heat-aggregated IgM. Results showed that 28.9% (SD +/- 5.1) and 5.3% (SD +/- 2.2) of the cells bear Fcnu or Fc mu receptors, respectively. The specificity of the receptors was tested after incubation of the cells in the presence of the following fluorochrome-conjugated reagents: non-aggregated Xenopus Igs and human IgG, aggregated Xenopus albumin, Fc6 nu and Fab mu fragments and human IgG, and antigen-complex Fab2 nu fragments. Results indicated that the receptors are specific for Xenopus immunoglobulins, with the restriction that the latter must be presented in a complexed or aggregated form to the cells and that the precise binding site is located on the Fc portion of IgY or IgM. The identity of a large percentage (22.4 +/- 2.8%) of cells bearing Fc nu R was established by direct simultaneous double-fluorescent staining of surface membrane IgM and Fc nu R. All Xenopus splenic B lymphocytes bearing sIgM carry also Fc nu R, while 8.8% of splenocytes, of yet unknown lineage, bear Fc nu R alone on their surface. The presence of Fc receptors on Xenopus B lymphocytes (leaving aside their eventual presence in other leucocyte types) suggests, once again, a high degree of evolution of the immune system in these lower vertebrates.     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.     

Expression of Fc epsilon receptors on activated human T lymphocytes

Our results clearly demonstrate that the low-affinity receptor for IgE (Fc epsilon R) is an activation antigen transiently expressed on a subpopulation of human T lymphocytes. It can be selectively induced by stimulation with certain antigens or lectins, but it is not found on resting T cells. The increased numbers of activated Fc epsilon R+ T cells observed after stimulation of peripheral blood mononuclear cells (PBMC) from bee venom allergic patients with the specific allergen phospholipase A2 (PLA2) suggest that Fc epsilon R+ T cells might very well be involved in the regulation of the human IgE response against the respective antigen. These results were obtained by the use of two monoclonal antibodies, M-L25 and M-L47, which were raised against the human low-affinity Fc epsilon R in our laboratory. After stimulation of PBMC with phytohemagglutinin a peak of 7.6 +/- 6% Fc epsilon R+ T cells was observed on day 3, with pokeweed mitogen of 0.8 +/- 0.8% on days 2 and 3, and with concanavalin A of 0.6 +/- 0.7% Fc epsilon R+ T cells on day 2. Stimulation of PBMC with tetanus toxoid (TT) induced Fc epsilon R on maximally 0.6 +/- 0.8% of the total T cells (day 4), stimulation with purified protein derivative from tuberculin (PPD) on 0.2 +/- 0.6% of the T cells (day 2). In contrast to these antigens, stimulation of PBMC from bee venom allergic patients with PLA2 induced as a peak 2.5 +/- 2.5% of the total T cells to express Fc epsilon R (day 5), although the stimulated T cell population was much smaller than with TT or PPD, as was shown by their stimulation indices. The allergen-stimulated Fc epsilon R+ T cells were exclusively T4+. The Fc epsilon R-expression index was determined, which for a specific antigen or lectin correlates the percentage of Fc epsilon R+ T cells to the stimulated T cell population, respectively.     

Antibody penetration into living cells. I. Intranuclear immunoglobulin in peripheral blood mononuclear cells in mixed connective tissue disease and systemic lupus erythematosus.

We have shown recently (Alarcón-Segovia, Ruíz-Argüelles & Fishbein, 1978) that an IgG anti-RNP antibody obtained from a patient with mixed connective tissue disease (MCTD) can penetrate viable mononuclear cells (MNC) from normal donors via their Fc receptors. Live MNC from twelve MCTD patients incubated with goat anti-Ig antibody had intranuclear antibody with a speckled pattern in a mean of 5.5% of all MNC and 57.3% of all Fc receptor-bearing MNC. We found intranuclear immunoglobulins in all twelve patients with MCTD which were present only in cells with Fc receptors. Only three out of twenty-one patients with systemic lupus erythematosus (SLE) were found to have intranuclear antibody in a mean of 17.2% of their Fc receptor-bearing cells. Further experiments with MNC from SLE patients revealed a partial blocking of penetration of antibody via Fc receptors. MNC from ten scleroderma, ten rheumatoid arthritis patients and eleven normal controls did not have intranuclear immunoglobulin. In vivo penetration of autoantibodies into Fc receptor-bearing cells in MCTD, and probably in SLE as well, may represent an important pathogenetic mechanism.     

Identification and distribution of immunocompetent cells in inflamed gingiva of human chronic periodontitis

Advanced human periodontitis is considered to be a B-cell lesion, but the cellular infiltrate contains several cell types, the distribution of which has not been determined. This experiment was designed to characterize and identify the immunocompetent cells on histological sections and in eluates from diseased human gingiva. Immunoglobulin-bearing cells were detected on histological sections by direct immunofluorescence with F(ab')2 antisera monospecific for human immunoglobulin G (IgG), IgA, or IgM. Plasma cells predominated in the central portion of the lamina propria, with the proportions positive for IgG, IgA, and IgM accounting for 65.2 +/- 9.5, 11.2 +/- 1.1, and 1.3 +/- 1.1% of the total infiltrating cells, respectively. T lymphocytes, identified by indirect immunofluorescence with monoclonal antibody (Leu-1) against human T cells, accounted for 29.3 +/- 10.0% of the total infiltrated cells. Most of the T cells were located subjacent to the pocket epithelium, but there were a few in the central lamina propria. Similarly, Fc receptor-bearing cells detected by EA rosetting and macrophages and monocytes detected by nonspecific esterase staining with alpha-naphthylbutyrate esterase were also localized to the region immediately subjacent to the pocket epithelium. Infiltrated cells were harvested from minced gingival tissue after digestion with collagenase, hyaluronidase, and DNase. The eluates contained 35.3 +/- 6.0% T lymphocytes, 30.0 +/- 14.9% Fc receptor-bearing cells, and 12.9 +/- 4.4% monocytes and macrophages. Whereas T gamma cells comprised 13.3 +/- 1.4% of peripheral blood T cells, they accounted for only 6.0 +/- 2.0% of the eluate T cells. In contrast, T mu cells accounted for 44.7 +/- 4.9% of the T cells in the eluates and 51.6 +/- 4.4% in the peripheral blood. The decreased proportion of T gamma cells in the gingiva may indicate a form of abnormal immune regulation concerned with T suppression of B-cell proliferation.     

Antibody penetration into living cells. IV. Different effects of anti-native DNA and anti-ribonucleoprotein IgG on the cell cycle of activated T gamma cells

Normal T cells bearing receptors for the Fc portion of IgG that were incubated in anti-RNP or anti-DNA at the time of activation with phytohaemagglutinin showed different effects on this activation as determined by flow cytometric analysis of acridine orange stained cells. Incubation in anti-RNP caused an arrest in the progression from the G0 + G1 to the S + G2 phases of the cell cycle. Incubation in anti-native DNA caused activated cells to have an increase in their RNA content without a concomitant increase in their DNA content (DNA block). These effects were not seen in T cells that were depleted of T gamma cells by means of their property of forming rosettes with high affinity for sheep erythrocytes. Use of F(ab')2 fragments of either autoantibody, pre-incubation with aggregated IgG, or incubation with the respective autoantibodies in the cold effectively prevented their effect on the nucleic acid content of T gamma cells. Despite their different effect on the cell cycle both antibodies caused similar increase of 51Cr release of low affinity T cells 6 h after incubation in them. Our findings show that different anti-nuclear antibodies seem to cause different effects upon the cells they penetrate. These differences may have pathogenetic significance in the diseases where these antibodies occur.     

Allergen-directed expression of Fc receptors for IgE (CD23) on human T lymphocytes is modulated by interleukin 4 and interferon-gamma

T lymphocytes bearing Fc receptors (FcR) for immunoglobulins are known to have immunoglobulin class-specific regulatory functions. Here we report that expression on T cells of the low-affinity FcR for IgE (Fc epsilon RII/CD23) is preferentially induced by stimulation with antigens that cause an IgE response. T cells from eight patients allergic to the hemoglobin of Chironomus thummi thummi mosquito larvae (CHIT I) were analyzed for reactivity with the anti-FcERII/CD23 monoclonal antibody (mAb) M-L25 under various conditions. No Fc epsilon RII/CD23+ T cells were observed among freshly isolated, resting peripheral blood mononuclear cells (PBMC). Stimulation of PBMC with CHIT I, however, induced a marked although transient Fc epsilon RII/CD23 expression on a large portion of the allergen-activated T lymphocytes. It reached a maximum of 37.2 +/- 4.6% Fc epsilon RII/CD23+ T cell blasts on day 5 of culture. The selectivity of this expression became evident when compared to non-allergenic control antigens: after stimulation of PBMC with tetanus toxoid or purified protein derivative from tuberculin a maximum of 4.6% +/- 1.4% and 4.2% +/- 1.1% T cell blasts was found to express Fc epsilon RII/CD23, respectively. Activation by an anti-CD3 mAb was insufficient to induce Fc epsilon RII/CD23 on T cells. The allergen-stimulated Fc epsilon RII/CD23+ T cells exclusively belonged to the CD4+CD29+ helper inducer T cell subset. Using a cDNA probe coding for the B cell Fc epsilon RII/CD23, Northern blot analysis revealed a 1.7-kb Fc epsilon RII/CD23 mRNA in extracts of highly purified allergen-stimulated T cells. It was of the same size as Fc epsilon RII/CD23 mRNA of the lymphoblastoid B cell line WI-L2. Of several cytokines tested [interleukin (IL) 1 to IL 6, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha] only IL 4 and IFN-gamma significantly modified allergen-induced Fc epsilon RII/CD23 expression on T cells. The latter was enhanced nearly twofold in the presence of IL 4, and was almost completely abrogated by IFN-gamma. IL 4, however, could not increase the number of Fc epsilon RII/CD23+ T lymphocytes either alone or in combination with an anti-CD3 mAb. Taken together, the selective induction of Fc epsilon RII/CD23 on T cells by allergen and its inclusion in the regulatory network of cytokines point to an important role of Fc epsilon RII/CD23+ T lymphocytes in the human IgE response.     

High avidity periventricular IgG-Fc receptor activity in human and rabbit brain

Periventricular tissues from human and rabbit brains were examined for receptors for the Fc domain of immunoglobulin G (Fc receptors). Fc receptors were demonstrated using antibody-coated erythrocytes (EIgG) and covalently cross-linked rabbit IgG. EIgG adhered specifically to nonneuronal periventricular glial areas and was inhibited by IgG and Fc fragments, but not by Fab fragments or albumin. EIgG adherence was 50% inhibited with 6.7 X 10(-9) M IgG. This is 1/1000 the amount of IgG required to produce 50% inhibition of EIgG adherence to arachnoid FcR. Studies with covalently cross-linked IgG demonstrated a linear relationship between ligand size and inhibition of EIgG adherence. Uncoated erythrocytes, IgM-coated erythrocytes, or F(ab')2-coated erythrocytes failed to bind to periventricular tissue. Many nonspecific esterase-positive cells were found in the area of periventricular EIgG adherence; this esterase activity was sensitive to fluoride. These results provide, for the first time, evidence of inherent IgG receptor activity in the periventricular tissues, and suggest that the periventricular tissues play a protective role in the removal of IgG or IgG-antigen complexes which have entered the cerebral spinal fluid.     

IgE Fc receptor positive T and B lymphocytes in patients with the hyper IgE syndrome.

The percentages of peripheral blood lymphocytes (PBL), bearing Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) were determined in four patients with the hyper IgE syndrome by a rosette assay employing IgE and IgG coated fixed ox erythrocytes. The patients had 8 +/- 3% Fc epsilon R+ and 13 +/- 8% Fc gamma R+ PBL, compared to 1.2 +/- 1% Fc epsilon R+ and 17 +/- 4% Fc gamma R+ PBL for control donors. T cells were isolated by rosetting with neuraminidase treated sheep erythrocytes (EN). Indirect immunofluorescence with Lyt 3 monoclonal antibody (MoAb) to the sheep erythrocyte receptor, followed by rosetting for Fc epsilon R and Fc gamma R showed that the patients' T cells contained less than 0.1% Fc epsilon R+ and 1.4 +/- 0.2% Fc gamma R+ cells; T cells from the control subjects contained less than 0.1% Fc epsilon R+ and 11 +/- 4% Fc gamma R+ cells. The non-T (EN rosette depleted) cells of the patients included 56 +/- 18% sIgM+/sIgD+, 45 +/- 9% Fc epsilon R+ and 35 +/- 27% Fc gamma R+ cells. Indirect immunofluorescence with MoAb to IgM, IgD, and NK cells (antibody B73.1) followed by rosetting for Fc epsilon R and Fc gamma R, indicated that 92 +/- 2% of the Fc epsilon R+ cells and 9 +/- 7% of the Fc gamma R+ cells were B cells (mu+/delta+), while 3 +/- 4% of the Fc epsilon R+ and 30 +/- 23% of the Fc gamma R+ cells were NK cells (B73.1+). Thus, most of the Fc epsilon R+ non-T cells were B cells, and only a small fraction appeared to be NK cells. On the other hand, Fc gamma R+ B cells were outnumbered by Fc gamma R+ NK cells (B73.1+) by three to one. The data indicate that patients with the hyper IgE syndrome have increased numbers of Fc gamma R+ PBL, most of them being B cells, whereas their T cells contain less than 0.1% Fc epsilon R+ cells.     

Interaction between streptococcal IgG Fc receptors and human and rabbit IgG domains.

Groups A, C and G streptococci were tested for their ability to bind 125I-labelled fragments of human and rabbit IgG in order to localize their sites of interaction with IgG domains. Among the Group A streptococci, strains with IgG Fc receptors bound 85% of the added IgG Fc fragments in the test systems, whereas these strains showed practically no reactivity with F(ab')2, Facb (F(ab')2 + C gamma 2 domains) or pFc' (C gamma 3 domains). The Group C and Group G strains bound 48-100% of IgG Fc, but could also bind up to 36% of the added F(ab')2 in accordance with a previously described 'alternative' Fab reactivity. However, unlabelled IgG F(ab')2 or Facb showed no, or only slight, inhibitory capacity for the binding of 125I-labelled IgG Fc to the C and G strains. Collectively, these results indicate that Groups A, C and G streptococci require both the C gamma 2 and C gamma 3 domains for interaction with IgG, and most probably also bind in the interface region between the C gamma 2 and C gamma 3 domains as has been shown for staphylococcal protein A.     

Production and characterization of human hybridoma monoclonal anti-Sm and anti-U1RNP antibodies spontaneously produced from normal human lymphocytes.

A panel of human:human hybridomas secreting monoclonal anti-Sm/RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to the lymphoblastoid B cell line GM4672. The specificity of these antibodies was studied by direct binding and competitive inhibition in enzyme linked immunosorbent assays (ELISAs), and by immunoblotting. The stable subclones of these hybridoma monoclonal anti-Sm/RNP antibodies could be classified into four groups according to ELISA. Group I bound Sm/RNP only, group II bound Sm-RNP, Ro/SS-A and La/SS-B, group III bound Sm/RNP and ssDNA, and group IV bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. When antibodies from each of the groups were tested by immunoblotting, the following pattern of reactivity emerged. Group II and IV antibodies reacted with U1RNP-A, Sm-B'/B, Sm-D and Sm-E proteins, as well as the Ro/SS-A and La/SS-B proteins. In contrast, group I and III antibodies did not bind to any individual protein components of Sm/RNP,Ro/SS-A or La/SS-B antigens, but recognized their conformational epitopes. These results, therefore, directly demonstrate for the first time that normal-derived B cells have the genetic potential, revealed here by somatic cell hybridization, to produce anti-Sm/RNP antibody responses which are ordinarily only associated with systemic lupus erythematosus (SLE) and related connective tissue diseases.     

Electron microscopy of Fc receptors on human lymphocytes.

The membrane receptor for Fc portions of IgG (FcR) was localized on the cell surface of humans lymphocytes by electron microscopy. The electron microscopic markers for FcR were soluble ferritin 7S anti-ferritin immune complexes prepared in forty times antigen excess than needed at equivalence. Fc receptors on the lymphocytes labelled at 0 degree in the presence of sodium azide were seen as discontinuous patches on the cell surface. In control experiments, no labelling was observed, which included lymphocytes treated with ferritin only or with F(ab')2 immune complexes as well as glutaraldehyde-fixed lymphocytes treated with 7S anti-ferritin immune complexes. The findings are discussed with relation to the widely accepted membrane fluidity model.     

Immune complexes in Takayasu''s arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.     

Interaction between herpes simplex type 1-induced Fc receptor and human and rabbit immunoglobulin G (IgG) domains.

Cells infected with herpes simplex virus type 1 (HSV-1) express a cell surface receptor able to bind to the Fc region of immunoglobulin G (IgG). The ability of HSV-1-infected cells to bind 125I-labelled human and rabbit IgG and IgG fragments was studied to localize the site of interaction to the C gamma 2 or C gamma 3 domains of IgG. 125I-labelled IgG and IgG Fc fragments consisting of C gamma 2 and C gamma 3 domains bound strongly to HSV-infected cells and did not bind to uninfected cells. In contrast, 125I-labelled F(ab')2, Facb [consisting of F(ab')2 and C gamma 2 domains] and pFc' (consisting of C gamma 3 domains) fragments did not bind to any of these cells. Unlabelled IgG and IgG Fc fragments inhibited the interaction between 125I-labelled rabbit IgG Fc and the HSV Fc receptor, whereas F(ab')2, Facb and pFc' fragments failed to inhibit this interaction. These data indicate that the HSV Fc receptor requires both the C gamma 2 and C gamma 3 domains for interaction with the IgG molecule analogous to the known interaction of protein A of Staphylococcus aureus, the Fc binding proteins of Group A, C and G streptococci, and certain human rheumatoid factors.     

Ro/SS-A- and La/SS-B-reactive B lymphocytes in peripheral blood of patients with Sjögren's syndrome

The aim of this study was to investigate the production of anti-Ro/SS-A and anti-La/SS-B antibodies in peripheral blood (PB) of patients with Sjögren's syndrome (SS). The ELISPOT method was performed to quantify the frequency of PB lymphocytes spontaneously secreting anti-Ro/SS-A and/or anti-La/SS-B antibodies. The total number of IgG-, IgA- and IgM-producing cells was also quantified. The recombinant Ro 52-kD, Ro 60-kD and La 48-kD proteins were used as target antigens. Three of 18 SS patients had PB lymphocytes secreting IgG antibodies against the recombinant Ro 52-kD protein. The same three patients had high serum titres of anti-Ro 52-kD antibodies. In addition, these patients were classified as having severe disease, and all three had focus scores of ≥ 8 in biopsies of the labial salivary glands (LSG). The correlation between the number of PB cells producing IgG antibodies against the recombinant Ro 52-kD protein and the focus score was significant (P < 0.01). The results indicate that only SS patients with severe disease and high degree of local inflammation in LSG have B cells producing anti-Ro/SS-A antibodies in PB. Thus, most of the spontaneous autoantibody production must take place in other body compartments, e.g. in exocrineglands and probably also in the lymphoid organs and/or other mucosal sites.     

Characterization of Fc receptors for IgE on human alveolar macrophages.

Human alveolar macrophages (aM phi) isolated from lung lavages performed during bronchoscopy and after surgical removal of pulmonary lobes were analysed for Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) by rosette assays. A mean+/-s.d. of 8.0+/-2.6% of aM phi formed rosettes with fixed ox erythrocytes coated with an IgE myeloma protein (Eo''-IgE). The Eo''-IgE rosettes were inhibited by two IgE myeloma proteins and by IgE Fc fragments but not by myeloma proteins of the other Ig classes or by IgE denatured by heating or reduction and alkylation. Fresh ox erythrocytes sensitized with rabbit IgG antibodies (EoA) formed rosettes with 64.1+/-20.3% of the aM phi. Peripheral blood monocytes formed 10.6+/-1.2% Eo''-IgE and 90.2+/-6.0% EoA rosettes. Incubation of the aM phi with a goat antiserum to human lymphocyte Fc epsilon R inhibited Eo''-IgE rosette formation on aM phi by 80% but did not affect the percentage of EoA rosettes. The antiserum also inhibited Eo''-IgE rosettes formed by peripheral blood monocytes and cultured macrophage-like U937 cells but not those formed by basophilic granulocytes obtained from a patient with chronic myelogenous leukaemia. There was no relationship between age, sex, diagnosis or smoking history of the patients and the percentage of aM phi forming Eo''-IgE rosettes. These studies demonstrate that a subpopulation of human aM phi bear Fc epsilon R that share antigenic determinants with Fc epsilon R on lymphocytes and monocytes. Fc epsilon R(+) aM phi may play an important role in allergic and inflammatory pulmonary diseases by inducing the release of mediators of inflammation after interaction with IgE immune complexes.     

Presence of "La-like" antigens on human T lymphocytes bearing receptors for IgG.

Human peripheral blood T cells bearing receptors for the Fc portion of immunoglobulins were characterized by the formation of double rosettes with sheep red blood cells and IgG-coated chicken erythrocytes (EA). Treatment of cells by anti-"Ia-like" antisera inhibits the binding of EA. Inihbition appeared to be specific since anti-HLA-A and HLA-B antibodies did not inhibit the formation of EA rosettes, whereas F (ab')2 fragments of anti-Ia-like IgG were found to be as potent inhibitors as the whole IgG. This result shows that in human and in mice, Ia-like determinants arepresent, close to Fc gamma receptors, on the surface of the T lymphocytes bearing such receptors.