Inhibition Of Rosette Formation Research Articles

Structure and function of immunoglobulin domains. VIII. An analysis of the structural requirements in human IgG1 for binding to the Fc receptor of human monocytes.

A human mononuclear cell population, enriched with monocytes by adhering them to microexudate on Petri dishes previously used for fibroblast cultures was used in a homologous human system to form EA rosettes. IgG1, Fc, and subfragments of Fc representing the C gamma 2 and C gamma 3 domains were tested for their ability to inhibit rosette formation. Although Fc and IgG were equally effective in inhibiting rosette formation over the concentration range 10(-6) to 10(-5) M, all subfragments were inactive at these concentrations. At 10-fold higher concentrations the C gamma 2 fragment was still inactive; however, both the C gamma 3 fragments, pFc' and at C gamma 3, did show significant activity at this higher level. Reduction and alkylation diminished the inhibitory capacity of IgG 10-fold but had a lesser effect on Fc. In a parallel series of experiments the ability of IgG and Fc to inhibit granulocyte rosettes was found to be markedly diminished by reduction and alkylation in both cases. These experiments reveal differences between Fc receptors in different cells, confirm a role for the C gamma 3 homology region in monocyte Fc receptor recognition, but do suggest a requirement, either direct or indirect, for the C gamma 2 domain.

Fc receptors on rabbit lymphocytes. Existence of receptors for IgG antibody complexed with antigen; conditions for its detection.

The presence of Fc receptors (FcR) on rabbit peripheral blood leukocytes is demonstrated using rosette formation with an ox erythrocyte-antibody (EoxA) complex. The receptor is specific for the Fc fragment of IgG (neither IgM nor F (ab')2 anti-Eox mediates rosette formation) that is antigen-bound (aggregated rabbit IgG inhibits the rosette formation only transiently). The receptor is species-specific: guinea pig IgG/Eox, goat IgG/Eox and sheep IgG/Eox complexes do not show rosette formation, and goat IgG aggregates do not inhibit rosette formation. The origin of the target erythrocytes is of importance. Sheep erythrocytes are not useful, and within Eox large differences between donors were found. Rosette formation was only inhibited by pretreatment of the rosette-forming cells with homologous immune complexes, whereas the size of the antigen greatly influenced the degree of inhibition. The rabbit FcR is pronase-resistant, unlike the human and murine RcR. The interaction of IgG and the FcR is not inhibited by isolated C gamma 3 domains. Further evidence for the requirement of the whole Fc region was obtained in experiments where inhibition of the rosette formation was observed using antisera directed to the C gamma 3 and the C gamma 2 domain, respectively. Anti-Fab antiserum did not inhibit rosette formation. Results are discussed in relation to the mechanism of allotypic suppression.

Anti-self receptors. I. Direct detection of H-2L region-restricted receptors on murine thymocytes.

A high proportion (20--50%) of murine thymocytes form rosettes with either syngeneic or allogeneic erythrocytes. The specificity of this interaction was investigated by measuring the ability of different erythrocyte sonicates to inhibit rosette formation. With erythrocyte sonicates from recombinant mouse strains it was demonstrated that rosetting with syngeneic erythrocytes was mediated by H-2L and/or H-2D region-restricted receptors. The specificity of autorosetting was directly mapped to the H-2L region by the inability of erythrocyte sonicates from the BALB/c-H-2dm2 mutant, an H-2L-deletion mutant, to inhibit the rosetting of wild-type (BALB/c) thymocytes. The B10,2D2-H-2dm1 mutant, which has substantially modified H-2L and H-2D antigens, supported this conclusion. Furthermore, anti-H-2L sera were able to specifically block the inhibition of rosetting by erythrocyte sonicates. The above procedures clearly implicated the H-2L region in the thymocyte rosetting of d and k haplotypes. With the s haplotype the rosetting receptor was mapped to the H-2L/H-2D region, whereas with the b and q haplotypes rosetting was only mapped to the D end of the H-2 complex. This study also suggested complete cross-reaction between the thymocyte receptors carried by the k and d haplotypes, whereas the receptors of b, q, and s haplotypes were haplotype specific. In addition, the inhibition assay indicated that the rosetting of thymocytes with allogeneic and xenogeneic (rat) erythrocytes was mediated by a receptor primarily directed against self-H-2L. Finally, the critical role played by the H-2L region in this rosetting phenomenon was demonstrated by the inability of thymocytes from the H-2L-deletion mutant (H-2dm2) to rosette with syngeneic, allogeneic (rat) erythrocytes.

Lymphocytes bearing Fc receptors for IgE. IV. Formation of IgE-binding factor by rat T lymphocytes.

Culture of mesenteric lymph node cells from rats infected with Nippostrongylus brasiliensis resulted in the release of a soluble factor (IgE-binding factor) that can inhibit rosette formation of Fc epsilon R(+) lymphocytes with IgE-coated red cells. The factor is specifically absorbed with IgE-coated Sepharose. It has a m.w. of between 10,000 and 20,000. The major source of IgE-binding factor appears to be Fc epsilon R(+) T cells. The formation of IgE-binding factor by the lymphocytes was enhanced by IgE added to the culture medium. Evidence was obtained that Fc epsilon R(+) cells are involved in the induction of IgE-induced factor formation. Normal rat lymphocytes cultured alone failed to release IgE-binding factor, but incubation of normal lymphocytes with rat IgE resulted in the formation of IgE-binding factor and an increase in the proportion of Fc epsilon R(+) cells. It was also found that normal T cells formed the soluble factor upon incubation with IgE. In the induction of factor formation by normal lymphocytes, Fc gamma R(+) cells are essential; an Fc gamma R-depleted fraction failed to form IgE-binding factor upon incubation with IgE. The results suggest that interaction of IgE with Fc epsilon R(+) T cells and Fc gamma R(+) T cells induces the formation of IgE-binding factor(s).

Role of Binding Through C3b and IgG in Polymorphonuclear Neutrophil Function: Studies with Trypsin-Generated C3b

Abstract In previous studies of the role of IgG and C3b in opsonization, C3b has not been present on the target particle in the complete absence of other serum proteins. We have developed a system in which C3b alone is present on the particle, using trypsin cleavage of purified C3. Sheep erythrocytes (E) coated with trypsin-generated C3b (ETC3b) efficiently bound to but were not ingested by human neutrophils. Specificity of binding was demonstrated by inhibition of rosette formation with fluid phase C3 or C3b, and by a decrease in rosettes after treatment of ETC3b with C3b inactivator (C3bINA). Addition of IgG anti-E but not IgM anti-E to ETC3b markedly enhanced neutrophil binding and stimulated ingestion of these erythrocytes. There were minimal binding and ingestion of E coated with IgG alone. Binding of ETC3b to neutrophils was also insufficient to stimulate the release of superoxide anion (O2-), β-glucuronidase, and lysozyme. EIgG, prepared with increasing concentrations of IgG, promoted the release of O2- and granule enzymes in a concentration-dependent fashion, and ingestion was not required. Addition to ETC3b of a small quantity of IgG synergistically enhanced O2- release and degranulation. These results suggest that the Fc and C3b receptors on neutrophils have separate but complementary functions. C3b serves to promote efficient particle-cell attachment, thereby mediating contact between particle-bound IgG and the neutrophil's Fc receptor. This latter contact triggers ingestion and the intracellular events involved in microbial killing.

Appearance of rosette-forming macrophages in the lungs of influenza virus-infected mice.

Approximately one fifth of the macrophages obtained from the lungs of mice infected 2 to 5 days with influenza A/HK virus were found to rosette well with either unmodified human, chicken, or guinea pig erythrocytes, but not with erythrocytes from hamsters, sheep, or mice. Rosette-forming macrophages were seldom seen in suspensions from uninfected mice (3+/-3%) or mice infected 24 h previously (3+/-3%). Rosette formation was not due to virus hemadsorption, as indicated by the failure of specific antiserum to influenza virus to block rosette formation; by the induction of comparable levels of rosette-forming macrophages in the lungs of mice infected with herpes simplex virus type 2, a nonhemadsorbing virus; and by the inhibition of rosette formation at 4 degrees C. Instead, rosette formation appeared to be directly related to macrophage elicitation or activation since nonstimulated macrophage populations such as peripheral blood monocytes, macrophages from uninfected lungs, or noninduced peritoneal macrophages were not observed to rosette to any significant extent. Furthermore, peritoneal macrophages induced with filter-sterilized normal horse serum rosetted at levels comparable to that observed with cells from infected lungs. These results indicate that hemadsorption alone can not be used as a criterion of virus infection of macrophages. However, rosette formation may serve to identify macrophage subpopulations which are active in host defense against viral infections.

Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.

To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.

The Effect of Sulfhydryl and Amino Group Reagents on Human Lymphocyte – Sheep Erythrocyte Rosettes

We have studied the influence of certain chemical groups, located on the surface of lymphocytes or sheep red blood cells, on the ability of these cells to form spontaneous rosettes. We found that fluorescamine, which reacts with amino groups, inhibits rosette formation, while p-chloromercuriphenylsulfonic acid, which binds to sulfydryl groups, increases the percentage of the rosette-forming lymphocytes. The possible mechanism of action of the above reagents is discussed.

IgA-Fc Receptors on Mouse Lymphoid Cells

Abstract Receptors specific for IgA were sought on mouse lymphoid cells by using a rosetting technique in which cells are exposed to modified ox red blood cells (ORBC) coated with IgA myeloma proteins having specificity for the modified ORBC. It was found that 4 to 6% of unseparated spleen lymphoid cells (pre-incubated at 37°C overnight) formed rosettes with MOPC 315-coated TNP-ORBC, with QUPC 52-coated dextran-ORBC, and with SAPC 10-coated arabinogalactan ORBC, whereas modified ORBC alone or modified ORBC mixed with irrelevant myelomas did not form appreciable numbers of rosettes; the cells forming rosettes were not neutrophils as shown by appropriate staining techniques. Thirteen to 22% of spleen cells also formed rosettes with IgG-coated ORBC (either IgG rabbit anti-TNP-coated TNP-ORBC or rabbit IgG anti-ORBC-coated ORBC); however, by using similar techniques, no cells forming rosettes with IgM-coated ORBC could be found. The specificity of the IgA-binding sites thus demonstrated was shown in inhibition studies: IgA antigen-antibody complexes formed near equivalence or, indeed, purified IgA were found to inhibit rosette formation with IgA-coated ORBC, whereas IgG complexes or purified IgG were found to cause no such inhibition. These inhibition studies were buttressed by depletion studies in which removal of cells binding IgG-coated ORBC with rosetting procedures was shown to have little or no effect on the percentage of cells binding IgA-coated ORBC in the residual cell population. The cells capable of binding IgA-coated ORBC are at least in part T cells in that the binding of IgA-coated ORBC is a property of nylon-column passed cells containing <3% sIg positive cells; in addition, 2 to 3% of thymocytes bind IgA-coated ORBC. Finally, the IgA binding sites appear to be Fc-specific in that F(ab′)2 fragments of IgA do not block IgA-rosette formation. In summary, we have identified an IgA-Fc-specific binding site on mouse lymphoid cells, including mouse T cells.

Detection of isoimmune antibodies against a population of human B-lymphocytes

Rosette-forming ability of human lymphocytes was tested before and after their sensitization with isoimmune sera against histocompatibility antigens. Anti-HLA sera inhibited the ability of T- and B- lymphocyteè to form rosettes. Some sera inhibited rosette formation mostly of B-lymphocytes; they also possessed marked capacity to react with B-lymphocytes population in prolonged lymphotoxic tests.

Inhibition of E-rosette formation by two iron salts

The effects of four different metal salts on E-rosette formation by human peripheral blood lymphocytes and cells from a human T-cell line were examined. Pretreatment of lymphocytes with FeCl 3 and Fe-citrate inhibited rosette formation. The inhibition was related to cell and iron salt concentrations. Zinc chloride and Na-citrate had no significant effect on rosette formation. The results indicate that lymphocytes can bind Fe 3+ and the possible implications of this finding are discussed in relation to the known roles played by T lymphocytes in the control of erythropoiesis and cellular immunity.

Structure and Function of Immunoglobulin Domains

Abstract The ability of various fragments of human myeloma IgG1 to inhibit rosette formation between human anti-D-coated human red blood cells and human polymorphonuclear leukocytes has been investigated. Although IgG and Fc showed a dose-dependent inhibition of rosettes at equimolar concentrations, neither of the fragments corresponding to the Cγ2 and Cγ3 homology regions obtained by acid-tryptic cleavage of Fc was able to inhibit rosette formation. The pepsin fragment of Fc, pFc′, which represents the complete Cγ3 domain, was also unable to prevent rosette formation. Reduction and alkylation of IgG or Fc markedly diminished cytophilic activity as measured by this system. These data indicate that the site in human IgG1 bound by granulocytes is dependent on the full quaternary structure of Fc, a requirement in marked contrast to that noted for binding by macrophages and monocytes.

Receptors for sheep red blood cells on human B lymphoid WI-L2 cells

On the cultured human lymphoid cells WI-L2, which carry the traditional B cell markers—immunoglobulins, receptors for the Fc portion of IgG, and receptors for monkey red blood cells—the presence of T cell receptors was shown by the following points of evidence. (i) WI-L2 cells form rosettes with sheep red blood cells (SRBC), especially those treated with the sulfhydryl compound 2-aminoethylisothiouronium bromide or with neuraminidase. (ii) They specifically bind antibodies that block the rosette formation between T lymphocytes and SRBC. (iii) In rabbits, WI-L2 cells elicit the formation of antibodies which, after extensive absorption with B lymphoid cells, inhibit the rosette formation between T lymphocytes and SRBC and in conjunction with rabbit complement lyse only T lymphocytes. T-Cell receptors solubilized from WI-L2 cells by hypertonic salt extraction and partially purified by ultracentrifugation on a KBr gradient could not inhibit rosette formation between T lymphocytes and SRBC but did react with anti-T-cell receptor antibodies and were immunogenic.

Fc-rosette inhibition by pregnant women's sera and by rabbit anti-beta2-microglobulin.

Some anti-HLA sera showed Fc-rosette inhibitory activity, which had little correlation with type-specific anti-HLA activity. Cytotoxic titers did not correlate with Fc-rosette inhibition rate and these antisera showed considerable inhibition of rosette formation even after absorption of anti-HLA activity with T lymphocytes. These results suggest that some anti-HLA sera contain B-cell-specific inhibitory activity against Fc-receptor like anti-Ia antibody in mice. Rabbit antiserum against human beta2-microglobulin showed specific inhibition of Fc-rosette formation. It was suggested that Fc-receptor or Ia-like antigen in human has a close relation with beta2-microglobulin.

Inhibition of rosette formation by antithymocyte globulin

The peripheral blood leukocytes from 29 patients with adenocarcinoma of the colon were studied sequentially for the T-cell level, the rosette-inhibition titer of antithymocyte globulin and the blastogenic response to PHA and Con A to evaluate the T-cell immunocompetence. The level of E-rosette-forming cells and the blastogenic response did not reflect the immunocompetence of the T-cell population. Fluctuations in the rosette-inhibition titer of antithymocyte globulin (ATG) were observed; an increased ATG titer indicated an unfavorable clinical course, while a decreased ATG titer was observed with patients who had a favorable clinical course. The rosette-inhibition titer with antithymocyte globulin was observed to be an important indicator of competent T cells in patients with colorectal cancer.

Affinity labeling of dinitrophenyl specific immunoglobulin on the surface of murine myeloma cells

Murine myeloma cells possessing cell surface IgA molecules capable of binding the dinitrophenyl (DNP) hapten were used as a model system to test the feasibility of affinity-labeling antigen receptors. The reagent N-ϵ-(4- azido-2- nitrophenyl)- l- lysine was shown to have a higher affinity for cell surface receptor than either 2,4-dinitrophenyl-1-fluoride or 2,4-dinitrophenyl-1-azide as determined by inhibition of rosette formation between myeloma cells and trinitrophenyl substituted erythrocytes. The gross distribution of radiolabel in affinity-labeled cells indicated that intrinsic reactivity and lipid solubility of the labeling reagents were as important considerations as binding affinity in determining the most appropriate affinity-labeling reagent. When used at concentrations of 10 −7 M and less, all three reagents were capable of labeling IgA with some degree of specificity. However, labeling at sites other than the receptor occurred to a considerable extent indicating that this is a major problem in working with a system as heterogeneous as the cell surface.

Masking of Receptors for Sheep Erythrocytes on Human T-Lymphocytes by Sera From Breast Cancer Patients 2

Sera from 140 breast cancer patients and 38 controls were tested for their ability to inhibit sheep erythrocyte (E) rosette formation by normal, allogeneic lymphocytes. Inhibition of rosette formation by greater than 20% was found with 65% of stage I sera, 91% of stage II sera, 56% of stage III sera, and all stage IV sera. In contrast, only 13% of control sera was inhibitory. The inhibitory factor was found to bind to only a proportion of T-lymphocytes and could be removed from these lymphocytes by mild proteolytic digestion or extended washing. Examination of the properties of the inhibitory factor indicated that it differed from other substances that reportedly inhibited E-rosette formation.

Spontaneous rosette formation and rosette inhibition assays in patients with squamous cell carcinoma of the head and neck

Peripheral blood lymphocytes from patients with squamous cell carcinoma (SCC) of the head and neck were studied by spontaneous lymphocyte rosette and rosette inhibition (RI) assays prior to treatment. The patients were clinically staged and the results of the assays compared with the clinical stage of the disease. The percentage of T-lymphocytes as determined by the spontaneous lymphocyte rosette test was significantly lower (p less than .01) for the patient group when compared with a normal population. Patients with stage I and II disease did not differ significantly from controls. Individuals with stage III or IV disease, however, had significantly lower T-lymphocyte counts. The tumors were histologically graded as well, moderately well, or poorly differentiated SCC. Patients with poorly differentiated neoplasms had significantly lower T-cell counts. The RI assay (using horse anti-human thymocyte globulin to inhibit rosette formation) was abnormal in many of the patients but did not appear to be a more sensitive in vitro measure of cell mediated immunity in these patients. Performing both tests detected more patients with cellular immunologic incompetence than either one alone.

Modulation of Macrophage C3b Receptor Function by Cytochalasin-Sensitive Structures

A quantitative rosette assay was employed in order to determine if through pharmacologic probes we could gain an insight into the nature of the interaction between C3b-coated particles and the macrophage C3b receptor. Rabbit alveolar macrophage monolayers were challenged with chromium-labeled, complement-coated (via cold agglutinin) human erythrocytes (HEC3b) and the per cent of bound counts determined in the distilled water lysate. With this assay system in which ingestion is negligible, the cytochalasins (A greater than E greater than D greater than B) produced the most marked inhibition of rosette formation compared to control treated monolayers. No agent examined produced consistent augmentation. Cytochalasin A at 10(-5), 10(-6), and 10(-7) M inhibited rosette formation by 77+/- 2, 44 +/- 4 and 15 +/- 7 (S.E.), per cent, respectively. Cytochalasin E was also markedly inhibitory, Cytochalasins B and D produced approximately 30% inhibition at 10(-5) M. The cytochalasin effect was not secondary to an interaction between these agents and complement-sensitized erythrocytes, although cytochalasin E was also able to reduce erythrocyte-bouund C3b reactivity. Cytochalasin A and E modulation of the macrophage C3b reactivity occurred within a few minutes and was only slightly reversible. Cytochalasins A and E could also disrupt performed rosettes but the effect was not as pronounced as when these agents were present before and/or during the actual adherence phenomenon. Vinblastine and colchicine (10(-5) and 10(-6) M) also produced significant inhibition of rosette formation, although the magnitude of the effect was less than that for cytochalasins A and E. Further characterization of the vinblastine and colchicine effect demonstrated that the inhibition was rapid, irreversible over a 60-min incubation, and not explained by an alteration in macrophage attachment or in HEC3b reactivity. Agents producing insignificant inhibition of rosette formation included the following: dibutyryl cAMP and cAMP agonists (PGE1, theophylline), 8-bromo cGMP and cGMP agonists (carbachol, asorbic acid), dimethylsulfoxide, heparin, ethanol, dextran sulfate, DEAE-dextran, and poly-L-lysine. The data suggest that cytochalasin, vinblastine and colchicine sensitive membrane structures, most likely microfilaments and microtubules, are important in the interaction of C3b-coated particles with the macrophage C3b receptor.

A method for the detection and quantitation of Fc receptor sites on cells.

A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocytes coated with protein A of Staphylococcus aureus (ES) to form rosettes with cells treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and binds to a trypsin-resistant but pronase-sensitive receptor (considered an Fc receptor). The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc receptors/cell). The sensitivity of the method permits quantitation of less than 10(5) IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.