Enhanced Susceptibility of Melanocytes to Different Immunologic Effector Mechanisms In Vitro: Potential Mechanisms for Postinflammatory Hypopigmentation and Vitiligo

In postinflammatory hypopigmentation and in vitiligo, one observes histologic evidence of melanocyte damage, disappearance of melanocytes, and clinical loss of pigmentation. In the case of vitiligo, this loss of pigment is complete. There is considerable evidence that melanocytes are highly susceptible to autocytotoxic damage and perhaps to specific immunologic damage. We directly compared the susceptibility of cultures of melanocytes (M), keratinocytes (K), endothelial cells (EC), and fibroblasts (F) to hydrogen peroxide damage across a Wide range of concentrations (10^-7-10^-2 M) and analyzed the differences by computerized Probit analysis. Cytotoxicity was measured by three dye techniques: acridine orange/ethidium bromide (AO/EB), fluorescein diacetate (FD), and nigrosin (N). All three assays produced similar results. The order of susceptibility to H₂0₂ was M < EC < K < F. The LD₅₀ of melanocyte targets was two orders of magnitude lower than that of fibroblasts. The AO/EB assay was used to study immunologic cytotoxicity of melanocytes in the presence of sera from vitiligo patients plus either complement or cellular effectors of antibody-dependent cellular cytotoxicity (ADCC). Eleven vitiligo sera and 11 control sera were contrasted in 4- and 16-hr cytotoxicity assays. Vitiligo patients' sera containing antimelanocyte antibodies induced both complement lysis and ADCC of melanocytes. Thus the melanocyte is highly susceptible to peroxide-induced damage, complement lysis, and ADCC. In addition, antibodies in vitiligo sera appear to be an important trigger of melanocyte damage by complement and ADCC effectors and are likely to be involved in the melanocyte damage observed in vitiligo.     

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.     

Spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) in peripheral blood and draining lymph nodes of patients with mammary carcinoma

Summary In 14 patients with primary invasive mammary carcinoma (T_1–3N_0–1M₀) lymphocyte preparations obtained from peripheral blood (PBL) and tumor-free or metastatic lymph nodes (LNC) were examined for spontaneous (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) against the allogeneic melanoma cell line IGR3 and a thymoma cell line THY. The cytotoxic activities were compared with those of PBL from healthy women and of LNC from normal mesenteric lymph nodes. In addition, the percentages of E-, EA-, EAC-rosette-forming cells and of surface Ig (SIg)-positive cells were determined for all PBL and LNC suspensions tested.As a rule, LNC exhibited significantly lower SCMC and ADCC than the corresponding PBL preparations. The difference was particularly pronounced in ADCC assays, due to a strikingly low K-cell activity of LNC cells. Consonant with this observation was a reduced percentage of Fc-receptor-bearing cells in LNC suspensions. In SCMC assays the IRG3 targets used in three tests appeared to be less susceptible to LNC effectors than THY targets. No difference in cytotoxicity was noted between PBL from breast cancer patients and from normal women; nor did LNC from tumor-free or metastatic axillary nodes and normal LNC from mesenteric nodes show a significantly different degree of SCMC and ADCC. Abbreviations used in this paper: ADCC, antibody dependent cellular cytotoxicity; E, neuraminidase treated sheep red blood cells; EAox, ox erythrocytes sensitized with rabbit-anti-ox IgG; EAh, AB erythrocytes sensitized with anti M and N antiserum; EAC, ox erythrocytes sensitized with rabbit-anti-ox IgM and sublytic human complement; LNC, lymph node cells; MEM-FCS, minimal essential medium supplemented with heated fetal calf serum, antibiotics, etc.; NK, de]natural killing; PBL, peripheral blood lymphocytes; PBS, phosphate-buffered saline; SIg, surface immunoglobulin; SCMC, spontaneous cell-mediated cytotoxicity     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

Interferon and natural killing of human lymphoma cell lines after induction of the Epstein Barr viral cycle by superinfection

Interferon (IFN) production during natural killer (NK) cell assays with Raji, an EBV-carrying human lymphoma-derived cell line, was studied to determine whether IFN generated by effectors in vitro acted in target cell lysis. In 4-hr tests, Raji is insensitive to NK but becomes susceptible after superinfection with the P3HR-1 strain of EBV. IFN was not detectable by bioassay in supernatants from 4-hr assays, and the addition of antibody to IFN did not prevent the lysis of the superinfected Raji cells. In 18-hr tests the NK sensitivity of the superinfected Raji cells was markedly elevated, and a percent of the normal Raji cells was also killed. IFN alpha was found in supernatants from 18-hr tests. Antibody to IFN alpha markedly reduced the killing of superinfected Raji and slightly reduced cytotoxicity against control Raji in 18-hr tests. Taken together these results indicate that what is referred to as natural killing has IFN-related and IFN-nonrelated components.     

Human polymorphonuclear leukocyte-mediated cytotoxicity against varicella-zoster virus-infected fibroblasts

Polymorphonuclear leukocytes (PMN) were studied for their ability to mediate cytotoxicity against varicella-zoster virus (VZV)-infected and uninfected human fibroblasts in 51Cr release assays. PMN were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) against VZV-infected targets. Maximal ADCC was obtained with effector-to-target ratios of 100:1 and 18 h of incubation. Percent 51Cr release for 26 normal adults was 14.1 +/- 0.6 (mean +/- standard error) in the presence of pooled human seropositive sera (final dilution, 1:100) and 0.5 +/- 0.6 in the presence of pooled human seronegative sera. Addition of phorbol myristate acetate (PMA) enhanced PMN-mediated cytotoxicity against VZV-infected and uninfected targets. PMA-stimulated cytotoxicity was optimal with PMA concentrations of 200 ng/ml and effector-to-target ratios of 10:1, and antibody was not required; killing was detected as early as 3 h after incubation and was maximal after 18 h. Highly purified PMN were capable of mediating both ADCC and PMA-stimulated lysis. Catalase completely inhibited PMA-stimulated PMN cytotoxicity, but had no effect on PMN-mediated ADCC. PMN from patients with chronic granulomatous disease were capable of mediating ADCC, but not PMA-stimulated killing, against VZV-infected targets. Thus, PMN could kill VZV-infected targets by two different mechanisms: ADCC, which required antibody but not hydrogen peroxide (H2O2), and PMA-stimulated cytotoxicity, which required H2O2 but not antibody.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji

Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets.     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

Interferon and butyrate treatment leads to a decreased sensitivity of NK target cells to lysis by homologous but not by heterologous effector cells

Human K-562 and HHMS cells were pretreated with human recombinant interferon (IFN)-gamma and used as targets in NK assays against human and murine effector cells. A protective effect against NK lysis was observed only in the homologous assay, whereas no change or even a slight increase in NK sensitivity against heterologous effector cells was found. In cold target inhibition experiments IFN-treatment of K-562 cells led to a decrease in their capacity to act as competitors in the homologous NK assay, leaving their inhibitory capacity unaltered in the heterologous assay. In accordance with results observed using human NK targets, murine YAC-1 cells treated with mouse recombinant IFN-gamma did not lose their susceptibility to human NK cells. However, they were markedly less susceptible to lysis mediated by murine effectors. Butyrate, another compound causing decreased sensitivity of K-562 cells for human natural killing, also failed to reduce the susceptibility against murine NK cells. The results indicate that the NK-resistant tumor target phenotype caused by IFN or differentiation-inducing agents can only be detected by homologous but not by heterologous effector cells. This suggests that major differences exist between the inter- and intraspecies NK killing mechanisms.     

Treatment of a murine B cell lymphoma with monoclonal antibodies and IL 2

A transplantable murine B cell lymphoma was used to study combination therapy with anti-idiotype antibody and interleukin 2 (IL 2). Class-switched IgG2a and IgG2b antibodies were compared. A marked additive and sometimes synergistic effect was seen when IL 2 was combined with either IgG2a or IgG2b anti-idiotype antibodies. A synergistic effect was also seen when similar experiments were performed in nude mice. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays showed that IL 2 enhanced antibody-mediated lysis by peritoneal cells exposed to IL 2 in vitro in a dose-related manner. Peritoneal cells harvested from mice treated in vivo with IL 2 contained an increased number of T cells and asialo GM+ natural killer cells, and also mediated enhanced ADCC. Depletion of natural killer cells with anti-asialo GM and complement resulted in a marked decrease in the antibody-dependent cytotoxicity mediated by these peritoneal cells. The mechanism of synergy between monoclonal antibody and IL 2 may be due to the direct or indirect activation of natural killer cells mediating ADCC.     

Natural killing in immunodeficient patients

Natural killing (NK) capacity was evaluated in peripheral blood mononuclear cells from 14 patients with well defined primary immunodeficiency disorders and compared with the activity of those cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays against antibody-coated erythrocyte (killed primarily by monocytes) and lymphoid or tumor targets (killed exclusively by lymphoid cells). A selective inability to lyse antibody-coated lymphocyte targets was observed with cells from patients with x-linked agammaglobulinemia, suggesting the involvement of either a different lymphocyte subpopulation or membrane receptor for NK and ADCC, or that a different functional susceptibility exists for the two types of killing. The only immunodeficiency state in which lymphocyte NK activity was found to be lacking was severe combined immunodediciency disease.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Spatial Heterogeneity and Peptide Availability Determine CTL Killing Efficiency In Vivo

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity.     

Isolation of functional subsets of human peripheral blood monocytes.

Monocytes were isolated by counterflow centrifugation of Ficoll-Hypaque separated peripheral blood mononuclear cells. The monocytes formed a bimodal volume distribution of "large" and "small" phagocytic esterase-positive, peroxidase-positive cells with peaks at 470 and 410 mu3, respectively. The large monocytes were predominately Fc receptor positive, and were able to lyse both sensitized human and chicken erythrocyte targets in ADCC assays, whereas the small monocytes were largely FcR negative and were inactive against sensitized human erythrocyte targets. However, ADCC against chicken erythrocyte targets was seen in some fractions containing small monocytes and was probably due to FcR+ lymphocytes (K cells) in those fractions. These experiments establish that monocytes are effectors of ADCC against both human and chicken erythrocyte targets and that the peripheral blood monocyte is heterogeneous in size, function, and surface receptor distribution.     

Two distinct mechanisms of cytotoxicity by porcine alveolar macrophages in antibody-dependent and immobilized immune complex-dependent cellular cytotoxicity

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.     

Antibody-dependent cellular cytotoxicity against murine leukemia viral antigens: studies with human lymphoblastoid cell lines and human peripheral lymphocytes as effector cells comparing rabbit, goat, and mouse antisera.

A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.     

Human leukocytes kill varicella-zoster virus-infected fibroblasts in the presence of murine monoclonal antibodies to virus-specific glycoproteins.

Seven murine monoclonal antibodies reacting with major glycoproteins of varicella-zoster virus were tested for functional activity in assays for antibody-dependent cellular cytotoxicity (ADCC) and antibody-plus-complement-mediated lysis. Human peripheral blood mononuclear cells killed varicella-zoster virus-infected fibroblasts in the presence of three of four monoclonal antibodies directed against gp98/62 and a single monoclonal antibody directed against gp118. Neither of two monoclonal antibodies directed against gp66 was able to mediate ADCC. In 18-h assays, adherent effector cells were more active than nonadherent effector cells in mediating ADCC. Adherent cells treated with anti-Leu-11b and complement retained their cytotoxic activity, suggesting that monocytes are responsible for most of the adherent-cell-mediated cytotoxicity. Both immunoglobulin G1 and G2a murine monoclonal antibodies were able to participate in ADCC. Of the two immunoglobulin G2a monoclonal antibodies tested, both of which reacted with gp98/62, only one mediated lysis in the presence of complement. These results indicate that some murine monoclonal antibodies against major glycoproteins of varicella-zoster virus have functional activity in cytotoxicity assays.     

Human monocyte cytotoxicity to tumor cells. I. Antibody-dependent cytotoxicity.

Recent investigations examining mononuclear cell antibody-dependent cell-mediated cytotoxicity against tumor cell lines suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. We have found, however, that in an allogeneic assay system, human monocyte monolayers as well as lymphocytes mediate substantial lysis of 51Cr-labeled antibody-coated CEM lymphoblast tumor cells. This cytotoxicity is temperature-dependent and rapid, with most 51Cr release occurring in the first 4 hr of co-incubation. Interaction between target cell-bound antibody and the monocyte Fc receptor is necessary as demonstrated by the marked fall in antibody-dependent cell-mediated cytotoxicity (ADCC) produced by staphylococcal protein A, high concentrations of nonspecific immunoglobulin, and dilution of the target cell antiserum. Morphologic and functional characteristics of the monocyte-monolayer preparations establish their relative purity (greater than 95%) and indicate that monocytes and not contaminating lymphocytes are responsible for tumor cell lysis. Furthermore, preincubation of monocyte and lymphocyte preparations with latex particles or low concentrations of immunoglobulin distinguished monocyte from lymphocyte ADCC. Thus, normal human monocytes have the capacity to carry out antibody-dependent cytotoxicity against nucleated malignant target cells.