Immunomodulatory effect of Listeria monocytogenes infection. II. Natural cell cytotoxicity in infected mice

The natural cell cytotoxicity of blood leukocytes, splenocytes and peritoneal cells derived from Balb/c mice being on day 1, 3, 5, 7, 9 of listeriosis to allogeneic fibroblast L 929 was examined. The intensity of cytotoxic reaction was assessed by the per cent of specific 51Cr release from target cells. To check if this reaction correlates with the level of monocyte-macrophage lineage cells present in the effector cells of if it depends on the development of delayed type hypersensitivity to Listeria antigen, Balb/c mice were infected with Listeria monocytogenes and simultaneously treated with ATS. It was found that Listeria monocytogenes infection in mice enhanced the natural cell cytotoxicity of tested effectors cells, particularly of peritoneal cells in animals being on the 5th-7th day of listeriosis. The treatment of mice with ATS which is known to inhibit the development of delayed type hypersensitivity to Listeria antigen, completely suppressed the stimulatory effect of this infection on the natural cell cytotoxicity to allogeneic fibroblasts. This finding suggests that the enhancement of natural cytotoxic activity of tested effector cells results from the action of activated macrophages cooperating with Listeria sensitized T lymphocytes.     

Characterization of pig lymphocyte subpopulations by adherence to nylon wool.

The property of adherence to nylon wool was used to separate and characterize pig blood lymphocyte subpopulations. Thymus-dependent null cells proved the least adherent lymphocytes, and B cells, bearing surface Ig or Fc receptor, the most adherent. sIg+ lymphocytes detected by immunofluorescence and by 51Cr release cytotoxicity showed similar frequency and adherence properties. Lymphocytes rosetting with SRBC in PBS or in dextran had similar adherence patterns and showed both non-adherent and adherent components. Lymphocytes bearing major histocompatibility complex (MHC) Ia-like antigens shown by eosin exclusion or 51Cr release cytotoxicity were enriched in the adherent fraction, but also occurred in significant numbers in the non-adherent fraction. Non-adherent Ia+ cells were predominantly sIg- and probably T cells, whereas the adherent Ia+ group included the sIg+ cells and some sIg- cells.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) in Aujeszky's disease

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) was studied using as targets⁵¹Cr-labelled Vero cells infected with the Bartha strain of Aujeszky's disease virus (ADV). Using hyperimmune anti-ADV serum to sensitize the targets, porcine leukocytes from dextran-sedimented blood were found to be efficient effector cells yielding maximal⁵¹Cr release by 16 hours. Whilst complement-dependent cytotoxic antibody could be demonstrated no enhancement of ADCC by complement was found. The sera of pigs vaccinated i.m. with Bartha virus were titrated in ADCC using leukocytes as effector cells and the results compared with those obtained by virus neutralization. ADCC proved to be a much more sensitive technique and might, therefore, provide the basis for a reliable diagnostic test.Partially purified lymphocytes and polymorphonuclear leukocytes from blood and peritoneal exudates, and macrophages from exudates were found to mediate ADCC with hyperimmune serum, but differences were observed in the efficiency and timing of their cytotoxic effects.With 6 Figures     

Antibodies against Fc receptors to aggregated IgG of mouse spleen cells: selective blocking of the receptors of splenocytes and peritoneal macrophages, and abolition of the phagocytosis enhancement produced by the opsonizing IgG antibodies.

The Fc receptors to aggregated IgG of mouse spleen cells were solubilized with Nonidet P-40, absorbed by immune precipitate, and the complex obtained was used to raise anti-Fc receptor antibodies in a rabbit. The antibody and its F(ab')2 fragment inhibit binding of heat-aggregated IgG with mouse spleen cells and peritoneal macrophages. When F(ab')2 from the anti-Fc receptor antibody was absorbed exhaustively with mouse peritoneal macrophages, it still partially inhibited the reaction between aggregated IgG and mouse spleen cells devoid of the adherent cells. These data indicate that the Fc receptors to aggregated IgG which are located on the surface of splenocytes and peritoneal macrophages carry both common and private antigenic determinants. It was also demonstrated that the pretreatment of the macrophages with Fab' from anti-Fc receptor antibody abolished completely the phagocytosis enhancement produced by the IgG opsonins.     

MHV S peplomer protein expressed by a recombinant vaccinia virus vector exhibits IgG Fc-receptor activity.

We have previously shown that cells infected with mouse hepatitis virus (MHV) bind rabbit, mouse, and rat IgG by the Fc portion of the IgG molecule. This Fc-binding activity appeared to be mediated by the MHV S protein. S protein could also be precipitated from MHV-infected cells by a monoclonal antibody directed against the murine Fc gamma receptor (Fc gamma R). To prove definitively that the S protein mediates Fc-binding activity, we have expressed the MHV S protein utilizing recombinant vaccinia viruses. The anti-Fc gamma R monoclonal antibody, 2.4G2, precipitated recombinant S protein in cells of murine, human, and rabbit origin. Since the anti-Fc receptor monoclonal antibody does not react with human and rabbit Fc receptors these results demonstrate that the epitope recognized by this antibody is carried on the MHV S protein and is not murine in origin. Examination of various MHV isolates and escape mutants failed to identify the precise sequences in S responsible for the molecular mimicry of the murine Fc gamma R. These data are consistent with the hypothesis that a previously identified region of similarity between the S protein and the Fc gamma R mediates this activity. The Fc binding activity of S was expressed on the cell surface, since MHV-JHM-infected cells, but not uninfected cells, formed rosettes with anti-sheep red blood cell (SRBC) antibody-coated SRBC. The anti-Fc gamma R monoclonal antibody neutralized MHV-JHM and inhibited syncytium formation induced by the MHV S protein.     

Antibody--antigen complex stimulated lysis of non-sensitized sheep red cells by human lymphocytes. I. Requirements for IgG complexes.

IgG antibody--antigen complexes stimulated lysis of non-sensitized sheep erythrocytes (SRBC) by normal human peripheral blood lymphocytes (PBL). Heat-aggregated human IgG, rabbit IgG-ovalbumin complexes and rabbit IgG-sensitized ox erythrocytes (ORBC) were effective in the induction of SRBC lysis by PBL. However, IgM-sensitized ORBC and IgM-complement-sensitized ORBC were ineffective. As only SRBC and not ORBC or chicken erythrocytes (CRBC) were lysed under identical experimental conditions, it is conceivable that the SRBC receptor present on the T cell is involved. Furthermore, 45% inhibition of lysis was obtained by pretreating the effector cells with anti-human thymocyte globulin (ATG) and complete inhibition was obtained by adding SRBC stroma to the reaction mixture. The requirement for the inclusion of IgG complexes and the absence of specific anti-target cell antibody distinguish this reaction from natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Immune killer T cells would not appear to be responsible as eight different donors were used and none of these were cytotoxic to SRBC in the absence of IgG complexes. The induction of this cytotoxic reaction appears to require the recognition and interaction by the effector cells of two separate molecular entities, i.e. the SRBC membrane by the T cell and the IgG Fc region by an IgG-Fc receptor-bearing cell.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Specifically immune mouse T-cells can destroy H-2 compatible murine target cells infected with herpes simplex virus types 1 or 2.

Lymphocytes from the spleen (SL) or peritoneal cavities (PL) of HSV-1- or HSV-2-immunized C 3 H mice expressed pronounced cytotoxicity, as measured by 51Cr release assay, against L cells experimentally infected with HSF-types 1 or 2 but not against uninfected L cells. HSV-1 or HSV-2 immune lymphocytes induced substantially more 51Cr release when L-cell targets were infected with homotypic virus compared to those infected with heterotypic virus. Inasmuch as the cytotoxicity of specifically immune C 3 H SL for HSV-infected L cells was selectively eliminated by treatment with AKR anti-theta C 3 H serum plus complement, the effector cells in present system were theta-bearing T cells. Evidence has been provided which indicates that specifically immune T cells express cytotoxicity exclusively directed against different HSV-infected target cells (mouse embryo cells or mouse peritoneal macrophages) which share H-2 antigens with the effector cells.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Antibody-dependent cellular cytotoxicity of Coxiella burnetii-infected J774 macrophage target cells.

Cell-mediated immunity is clearly the critical host defense mechanism against human Coxiella burnetii infection (Q fever); the role of specific antibody is unclear. By using a mouse macrophage tumor cell line, J774, persistently infected with C. burnetii phase I organisms, in a standard 51Cr-release cytotoxicity assay, we explored the possibility that antibody-dependent cellular cytotoxicity may be immune mechanism in Q fever. After 16 h of incubation in the presence of immune sera from Q fever hepatitis or endocarditis patients, nonimmune human peripheral blood effector cells specifically lysed infected J774 target cells; no 51Cr release was seen in the presence of nonimmune sera or uninfected target cells. An effector/target ratio of at least 5:1 was required, and monocytes were more efficient effector cells than lymphocytes. Cytotoxicity was blocked by preincubation of effector cells with purified aggregated human immunoglobulin G, indicating the role of Fc receptor-bearing effector cells. Two nonphagocytic lymphoid tumor cell targets, passively coated with C. burnetii, did not induce substantial immune-specific cytolysis, suggesting that bystander lysis does not explain the observation of specific lysis. Although antibody-dependent cellular cytotoxicity may participate in primary defense, alternatively, it may facilitate the dissemination of C. burnetii or surreptitiously participate in granuloma formation.     

Failure to detect killer cell activity in rabbits.

Rabbit lymphoid cells from spleen, peripheral blood, and peritoneal cavity lacked killer (K)-cell activity against cell lines of rabbit and human origin, including virus-infected human tumor cells. This lack of activity was not affected by antibody concentration, source of antibodies, effector/target cell ratio, or length of assay. Rabbit leukocytes, however, were capable of lysing antibody-coated chicken erythrocytes. Hamster leukocytes, serving as a known source of K cells, mediated antibody-dependent cellular cytotoxicity against all targets. EA-rosette assays and mixed effector cell competition tests suggested a deficiency in rabbit K-cell activity which is not a result of an inherent lack of Fc receptor-positive cells or of some suppressor mechanism operating in the rabbit cell populations. Our data support the concept that antibody-dependent cellular cytotoxicity may not be a significant in vivo immune mechanism in certain species.     

Inactivation of varicella zoster virus in vitro: effect of leukocytes and specific antibody.

Inactivation of varicella zoster virus in vitro by nonadherent, mononuclear peripheral blood leukocytes and antibody is described. When leukocytes and specific antibody were incubated with this virus, marked inactivation of the virus occurred. In contrast, leukocytes alone or with serum devoid of varicella zoster antibody caused only a small degree of inactivation of varicella zoster virus. The leukocytes involved appeared not to be monocyte-macrophages or T or B lymphocytes, and only minute amounts of specific antibody were required. We had found previously that leukocytes from unsensitized (varicella susceptible) as well as sensitized (varicella immune) donors could cause this reaction. We therefore propose that the reaction may be a form of antibody-dependent cellular cytotoxicity, as has been described for 51Cr release by lymphoid (K) cells for other herpesviruses.     

Functional studies on purified eosinophils and neutrophils from patients with Schistosoma mansoni infections.

Unpurified peripheral blood leucocytes or purified eosinophils and neutrophils from patients with schistosomiasis and from normal individuals were compared for their ability to interact with antibody coated schistosomula of Schistosoma mansoni. There was no difference in the ability of buffy coat cells or neutrophils from patients and from normal individuals to mediate antibody-dependent 51Cr release from labelled schistosomula. However, eosinophils from patients were significantly better than those from normal individuals in causing antibody-dependent 51Cr release. This enhanced activity of eosinophils from patients with schistosomiasis was found to correlate with the intensity of their infection as judged by faecal egg counts. Eosinophils from patients also contained a higher proportion of cells with detectable Fc receptors than those from normal individuals. It is suggested that the difference in the behaviour of eosinophils from patients and from normals may reflect an 'activated' state of these cells in the infected individuals.     

Modulation of Fc tau receptor expression and function in mouse peritoneal macrophages by ammonium metavanadate.

Resident peritoneal macrophages (PEM) harvested from female B6C3F1 mice given an intraperitoneal injection of ammonium metavanadate (2.5 or 10 mg V/kg), an equivalent amount of ammonium in the form of ammonium chloride, or sodium phosphate buffer (0.1 M, pH 7.2) every third day for 6 weeks, were subjected to flow cytometric analysis of Fc tau 2a and Fc tau 2b receptor expression, and photometric microassay to measure receptor mediated binding and phagocytosis of sheep red blood cells (SRBC). The NH4Cl and 10V groups showed 21.7 and 17.2% lower mean fluorescence channel (MFC) values and 7.1 and 5.9% lower values in percentage fluorescence-positive cells than the phosphate buffer control with respect to Fc tau 2a expression. For Fc tau 2b expression, the 10V group showed significantly (P less than 0.05) lower MFC (31.2%) and percentage fluorescence-positive cells (15.7%) than the phosphate buffer control. Though the four groups did not show a significant difference in Fc tau 2a mediated binding and phagocytosis of SRBC, the 10V group showed a significantly lower Fc tau 2b mediated binding and phagocytosis. The results indicate that the reduction in Fc tau 2b expression and function could contribute toward the previously observed depression in phagocytosis, NADPH-oxidase and superoxide generation in peritoneal macrophages obtained from vanadate-treated animals.     

Apparent direct cellular cytotoxicity mediated via cytophilic antibody. Multiple Fc receptor bearing effector cell populations mediating cytophilic antibody induced cytotoxicity

Guinea-pigs immunized with chicken red blood cells (CRBC) developed cytotoxic effector cells in peripheral blood, spleen, lymph nodes, bone marrow and peritoneal exudate cells. Although it appeared that direct cytotoxicity was the mechanism of killing in this model, the true mechanism of cytotoxicity was in fact cytophilic antibody firmly bound to the effector cell rendering it specifically cytotoxic to the CRBC targets. Using multiple cell separation procedures, we demonstrated at least three distinct effector cell populations capable of mediating cytotoxicity in this model: a monocyte-macrophage, a non-phagocytic lymphocyte and a neutrophil, all bearing Fc receptors for Ig. Cell free eluates produced from immune effector cells were capable of rendering non-immune cells of all three Fc receptor bearing leucocyte classes cytotoxic.It is noteworthy that several techniques commonly employed to deplete effector cell populations were shown also to remove cytophilic antibody from the surface of these effector cells. If this had not been recognized, the cytophilic antibody component of the system would have been overlooked and erroneous conclusions would have been made as to which cell populations were functioning as effectors.Recent clinical studies have demonstrated a direct cytotoxicity by K lymphocytes—the usual effector cells in antibody dependent cellular cytotoxicity. The present study suggests that in at least some of these cases true direct cytotoxicity may not be the mechanism of killing and that K cells bearing cytophilic antibody may in fact be the effector cell operating by antibody dependent cellular cytotoxicity.     

Wheat-germ agglutinin initiates monocytoid cell killing of non-antibody-coated erythrocytes

Cells bearing alien surface antigens can be specifically destroyed by antibodies directed to the antigens and certain Fc receptor-bearing lymphoid or monocytoid cells through a killing mechanism known as antibody-dependent cellular cytotoxicity (ADCC). The present study was undertaken to see if lectins could substitute for specific antibody in ADCC and initiate lysis of non-antibody-coated erythrocytes. Human peripheral blood leucocytes and mouse PU-5 monocytoid cell lines were used as effector cells. Target-cell destruction was measured by the specific release of 51Cr. Our data indicated that Wheat-germ agglutinin (WGA), but not concanavalin A or soybean agglutinin, could activate killing of non-antibody-coated erythrocytes by human peripheral blood leucocytes and PU-5 cells. The membrane structure on the effector cell that was triggered by WGA directly related to N-acetyl-glucosamine and may be adjacent to, if not part of, the Fc receptor.     

Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host.     

Immune reactions in human filariasis.

Sera from cases of elephantiasis due to Wuchereria bancrofti infection promoted an intense adhesion of peripheral blood leukocytes to W. bancrofti microfilariae in vitro. A similar adhesion was also seen using sera from some normal persons living for several years in areas where filariasis is endemic. No such adhesion was evident with sera from microfilaria carriers or from normal subjects from nonendemic areas. The adhesion was complement independent and was associated with the immunoglobulin G fraction of serum. 51Cr release studies suggested the occurrence of cell-mediated cytotoxicity to W. bancrofti microfilariae in the presence of elephantiasis serum. Microfilariae of Litomosoides carinii could be isolated free of blood cells, from the blood of infected rats. In the presence of serum, or its immunoglobulin G fraction, from patients with elephantiasis, L. carinii microfilariae adhered to human peripheral blood leukocytes or rat spleen cells.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. IV. Fc receptors on specific peritoneal exudate lymphocytes and their role in delayed-type hypersensitivity reactions.

In mice, delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) is mediated by T cells. Peritoneal exudate T cells (PETLs) from mice optimally sensitized for DTH to SRBC form rosettes when interacted with sensitized sheep red blood cells (EA). The binding of EA to PETLs is mediated by a receptor specific for the Fc portion of the antibody (FcR). Biological activity (mediation of DTH) depends on the unreacted state of PETLs and is lost when the latter are either rosetted with EA or reacted with aggregated IgG. Transfer of EA or aggregated IgG-treated PETLs from mice with DTH to SRBC does not lead to adoptive sensitization of recipients. It is suggested that FcR found on the membrane of T cells mediating DTH play a role in the regulation of the cellular immune response to SRBC.     

Increased IgE-dependent cytotoxicity by blood mononuclear cells of allergic patients.

Peripheral blood mononuclear cells from 14 healthy donors and 22 allergic patients were incubated with 51Cr-labelled chicken erythrocytes coated with an IgE myeloma protein or rabbit IgG antibodies. Mononuclear cells from patients with severe atopic disorders released a significantly greater percentage of 51Cr (P less than 0.001) from IgE-coated target cells than mononuclear cells from healthy controls, patients with mild atopic disease, or patients with severe atopic disease taking oral prednisone. Specific 51Cr-release from IgE-coated target cells was directly correlated to the percentage of monocytes (latex-ingesting cells) with Fc receptors for IgE (r = 0.87, P less than 0.01) as detected by a rosette assay employing ox erythrocytes coated with IgE. Mononuclear cells from patients and normals released similar amounts of 51Cr from IgG-sensitized target cells. Depletion of monocytes from mononuclear cell preparations from two severe atopic patients decreased 51Cr-release from IgE-coated target cells to levels seen in healthy donors or patients with mild allergic disease. These results demonstrate that mononuclear cells from severely allergic patients have a significantly increased cytotoxicity toward IgE-coated targets coated target cells and that this cytotoxicity correlates highly with the percentage of monocytes with Fc receptors for IgE in these mononuclear preparations.