Plaque-forming Cell Response Research Articles

Immunological studies on paroxetine, a novel anti-depressant drug

Paroxetine is a novel and selective neuronal 5-hydroxy-tryptamine uptake inhibitor with antidepressant activity. Paroxetine was examined for its ability to induce adverse immunological reactions, either as a consequence of a specific immune response or by a direct or indirect effect on the immune system. Paroxetine did not react in vitro with protein amino or thiol groups, suggesting that it lacks the capacity to form potentially immunogenic hapten protein conjugates. No anti-paroxetine antibody was detected in plasma or serum samples from patients and rats following oral administration over prolonged periods, or from epicutaneously exposed guinea pigs, or from rabbits given paroxetine in Freund's adjuvant, suggesting that paroxetine does not have the capacity to elicit humoral immune responses. Guinea pigs epicutaneously exposed to paroxetine did not develop contact sensitivity, suggesting that it does not have the capacity to elicit cell-mediated immune responses. These results suggest that paroxetine lacks intrinsic immunogenicity. Anti-SRBC antibody plaque-forming cell responses in mice were unaffected by oral administration of paroxetine, and paroxetine had no significant effect on ex vivo and in vitro murine macrophage phagocytosis of opsonized SRBC or on ex vivo murine splenocyte mitogen responses, suggesting that paroxetine does not exert modulatory effects on the immune system or on macrophage function. These findings, together with the results of pre-clinicsl safety evaluation studies, suggest that paroxetine is unlikely to have immunotoxic effects.

Immunosuppression in malaria: effect of hemozoin produced by Plasmodium berghei and Plasmodium falciparum.

To a considerable degree, malaria-induced immunosuppression has been attributed to an inhibition of macrophage accessory cell function. In this study hemozoin, a plasmodium hemoglobin degradation product which readily accumulates in phagocytic cells and tissues during infection, was examined for its influence on immune responses. Hemozoin-laden liver and splenic macrophages from Plasmodium berghei-infected mice, displayed accessory cell dysfunction which was likely due to hemozoin loading by these phagocytic cells. This indicated by the observation that hemozoin obtained from livers and spleens of infected mice as well as from Plasmodium falciparum cultures greatly inhibited splenic plaque-forming cell responses to sheep red blood cells. The results of the present study strongly suggest that the inhibition of macrophage accessory cell activity is due, at least in part, to the uptake and accumulation of hemozoin in their cytoplasms.

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro.

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.

The effect of cyclophosphamide on B cells and ‘background’ immunoglobulin-secreting cells in mice

The influence of cyclophosphamide (CY) was studied on the B-cell compartment of mice. This was done at five different levels: (a) the serum immunoglobulin (Ig) levels; (b) the numbers of ‘background’ Ig-secreting cells; (c) the incidence of surface Ig+ B cells; (d) the capacity of lipopolysaccharide-reactive B cells to give rise to a polyclonal IgM- and IgG-response in vitro; and (e) the capacity of long-lived memory B cells to give rise to an adoptive anti-sheep red blood cell plaque-forming cell response in vivo. A single injection of 300 mg CY/kg body weight (BW) decreased the numbers of background IgM-, IgG- and IgA-secreting cells in spleen, bone marrow and lymph nodes to minimum values of about 25% of normal at day 7. The incidence of surface Ig+ B cells also gradually decreased after CY treatment. The functional capacity of the B cells, however, was completely abolished one day after a single injection of 300 mg CY/kg BW. This was found for the lipopolysaccharide-reactive B cells, which largely represent newly formed, short-lived B cells as well as for long-lived memory B cells. The decrease of background Ig-secreting cells following a single injection of 300 mg CY/kg BW was followed by a gradual recovery with a substantial overshoot peaking about 40 days after CY injection. After multiple injections of 100 mg CY/kg BW, the minimum values of background Ig-secreting cells in the various lymphoid organs were lower than after a single injection of 300 mg CY/kg BW, but in this case the recovery was not associated with an overshoot reaction. Remarkably, the serum Ig levels were less profoundly affected than the numbers of Ig-secreting cells in the various lymphoid organs.

Modulation of the in vitro murine immune response by met-enkephalin

The in vitro priming of mouse spleen cultures with sheep erythrocytes (SE) was used to study the modulation of immune function by met-enkephalin (MENK). In these studies, suboptimal, optimal, and supraoptimal concentrations of SE were used to manipulate the plaque-forming cell (PFC) responses of cultured spleen cells. MENK, at a concentration of 10 −7 M, was able to abolish the high antigen dose-induced suppression of the PFC response, but was unable to increase the PFC response of cultures treated with suboptimal doses of antigen. On rare occasions when the supraoptimal dose of antigen did not suppress the immune response, the addition of 10 −7 M MENK to the culture medium suppressed the PFC response. Naloxone was unable to block the effect of MENK. These results indicate that the nature of the immune response must be taken into consideration when evaluating the effect of opioid peptides on immune function. We propose that MENK possesses a dual modulatory role, with the abilities to suppress a strong immune response and reverse high antigen-induced immunosuppression.

Low molecular weight factors displaying augmenting activity for human antibody production in vitro.

Dialyzable low molecular weight antibody-augmenting factors (LMAAF) were found in the culture supernatant of human tonsillar lymphocytes which were not stimulated by antigen and/or mitogen in vitro. Phagocyte-depleted nylon wool-adherent lymphocytes (M-Ny+ cells) were responsible for the release of the LMAAF. Marbrook's culture system was adopted to assay for the LMAAF. The M-Ny+ cells, which were cultured without antigen and/or without mitogen in the reservoir of Marbrook's diffusion culture vessel, released the LMAAF, which diffused across a dialysis membrane and significantly augmented the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) response of phagocyte-depleted lymphocytes (M-cells) cultured in the inner vessel. Phagocyte-depleted nylon wool-passed lymphocytes (M-Ny- cells) cultured in the reservoir could not augment the PWM-induced PFC response of the M- cells cultured in the inner vessel. The exuded fluid, which was the dialysate of the culture supernatant of the M-Ny+ cells ultrafiltrated with dialysis tubing, also enhanced the PFC response of M- cells cultured in 24-well multi plates. The exuded fluid also augmented the total IgM and IgG production of human tonsillar and peripheral blood lymphocytes measured by enzyme-linked immunosorbent assay (ELISA) systems. Gel filtration chromatography on Sephadex G-25 Superfine column showed that the LMAAF activity was demonstrated in the fractions corresponding to a molecular weight (m.w.) of 362 to 1,355 and a m.w. of 3,560 to 5,700, with a peak activity at about 4,500 dalton. The LMAAF were inactivated by treatment with proteinase K, but not by trypsin, alpha-chymotrypsin, RNase, and DNase, and were stable when treated at 56 C for 60 min. The dialysates of culture supernatants from two out of seven Epstein-Barr virus (EBV)-transformed M-Ny+ cell lines showed LMAAF-like activity. These results indicate that phagocyte-depleted nylon wool-adherent lymphocytes, possibly B cells, release low molecular weight factors displaying augmenting activity for human antibody production in vitro.

Mechanisms of specific immunological unresponsiveness to bacterial lipopolysaccharides.

Low-dose priming of mice with Escherichia coli O113 lipopolysaccharide (LPS) results in the development of immunological memory, whereas low-dose priming with E. coli O55 LPS or Serratia marcescens LPS induces significant antigen-specific unresponsiveness. All three preparations of LPS induced proliferation of mouse splenocytes with similar time course and [3H]thymidine uptake. There was no correlation between the small amounts of serum antibody detected by enzyme-linked immunosorbent assay after low-dose priming and the subsequent generation of either memory or unresponsiveness. Further, the passive transfer of small amounts of LPS-specific antibody had no significant effect on the magnitude of the plaque-forming cell (PFC) response elicited after subsequent immunization. Reduction of the PFC response to E. coli O55 LPS occurred after low-dose priming of nu/nu (as well as nu/+) mice; however, unresponsiveness could not be generated in nu/nu mice by low-dose priming with S. marcescens LPS. Thus, although the development of low-dose unresponsiveness to S. marcescens LPS appears to involve T cells, the response of E. coli O55 LPS does not. Enhancement of the primary PFC response to S. marcescens LPS could be transferred with low-dose primed spleen cells depleted of Lyt-2+ T cells; this suggests that the magnitude of the PFC response to this preparation of LPS is negatively influenced by Lyt-2+ T cells and positively influenced by Lyt-2- spleen cells (i.e., L3T4+ T cells). These findings indicate that T cells appear to be involved in regulating the magnitude of the antibody response to some types of bacterial LPS.

Prior exposure to subimmunogenic amounts of some bacterial lipopolysaccharides induces specific immunological unresponsiveness.

Pretreatment (priming) of BALB/c mice with a low (subimmunogenic) dose of Escherichia coli O113 lipopolysaccharide (LPS) generates immunological memory 7 to 30 days later; the direct (immunoglobulin M) plaque-forming cell (PFC) responses produced after subsequent immunization with an optimal dose are 4 to 20 times greater than those of unprimed mice. By contrast, priming with a low dose of E. coli O55 LPS, followed by immunization with an optimally immunogenic dose 2 to 30 days later, resulted in a significantly reduced antibody response. Similar results were obtained with Serratia marcescens LPS. Dose-response studies indicated that such unresponsiveness is antigen specific and could be induced with subimmunogenic amounts of LPS. Priming reduced the magnitude of the PFC response to all immunizing doses of LPS tested. Unresponsiveness is not due to (i) an alteration in the time course of the PFC response or to (ii) a change in the isotype of the anti-LPS antibody produced after priming and immunization.

Immunoregulatory abnormalities induced by experimental reovirus infection: Functional alterations in T-cell subpopulations

A primary anti-sheep red blood cell (SRBC) plaque assay system was used to analyze the effect of reovirus infection on immunoregulatory T-cells. A decrease in the plaqueforming cell (PFC) response of splenic lymphocytes was observed within 24 hr of infection with 10 9 or 10 11 particles of reovirus type 1 or reovirus type 3 and persisted for more than 7 days. In coculture experiments, T-cells from infected mice were found to produce less help to control B-cells and to suppress helper function mediated by control T-cells. This suppression did not require the presence of an Lyt1,2,3 cell. In addition, isolated Lyt1 cells from reovirus-inoculated mice provided less help than did Lyt1 cells from normal controls. These abnormalities were more marked following inoculation with reovirus type 3 than with type 1. Live virus was not required for these effects, as T-cells from mice inoculated with ultraviolet (uv)-inactivated reovirus when added to normal B-cells reproduced the effects of infection with live virus. Furthermore, these changes were not the result of alterations in the percentages or numbers of distinct Lyt-bearing T-cell subpopulations in the spleens of inoculated mice. Thus, humoral hyporesponsiveness following reovirus infection of adult mice is associated both with active T-cell suppression of B-cell help and with decreased help mediated by the Lyt1 subset.

Modulation of the anti-phosphorylcholine immune response duringTrichinella spiralisinfections in mice

The nematode Trichinella spiralis is able to modulate the antibody response, as measured by the plaque-forming cell (PFC) technique, to three thymus-dependent (TD) antigens: (1) a heterologous antigen unrelated to the parasite (sheep red blood cells (SRBC]; (2) an antigenic fraction, rich in phosphorylcholine (PC), obtained from T. spiralis (FCp1) and (3) a heterologous antigen unrelated to the parasite, but sharing the PC epitope with the FCp1 fraction (PC-KLH). During the life-cycle of the parasite in BCF1 mice, two opposing immunomodulating activities occur: (1) an immuno-potentiating activity in mice infected during the intestinal and larval migratory stages, for all three antigens, and (2) a carrier-specific immunosuppressive response in mice infected and immunized with the FCp1 fraction during the muscle phase of the life-cycle. The anti-PC PFC response of these mice is dependent on the infection dose and decreases from day 35 post-infection (p.i.) until at least day 85 p.i. The factor responsible for the stimulating effect observed during this stage is the presence of migratory larvae in the host. All the foregoing seems to indicate that T. spiralis can use specific suppression mechanisms to aid in its own survival.

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo- p-dioxin antagonist: Effects on enzyme induction and immunotoxicity

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD ⪢ Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effect of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 μmol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10 000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 μmol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 μmol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 μmol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

Immunoregulation in the Peyer's patch microenvironment. Cellular basis for the enhanced responses by the B cells of X-linked immunodeficient CBA/N mice.

The Peyer's patches (PP) of X-linked immunodeficient (xid) CBA/N and hemizygous (CBA/N X DBA/2)F1 (CDF1) male mice contain a B cell subpopulation that expresses the Lyb-5 maturational marker and is responsive to type 2 and T cell-dependent antigens in vitro, a B cell phenotype which is absent from the spleens of xid mice. Experiments reported here show that xid spleen B cells co-cultured with B cell-depleted PP cells from xid mice differentiated into specific plaque-forming cells in response to trinitrophenyl-Ficoll (type 2) and sheep erythrocytes (T cell-dependent). Two cell types were involved in this normalization of xid B cell responses. An accessory cell activity present in the PP, but not the spleens, of both CDF1 male (xid) and CDF1 female (normal) mice was required for the response to either the type 2 or T cell-dependent antigens. In the presence of this PP accessory cell, T cells from the PP of either xid or normal mice supported responses to both classes of antigens. In contrast, T cells from the spleens of xid mice did not support the response to trinitrophenyl-Ficoll, although the splenic T cells from normal mice did synergize with PP accessory cells in allowing plaque-forming cell development by xid B cells to this type 2 antigen. The xid PP T cell activity required for the type 2 response by xid B cells was present in the Ly-1+, Lyt-2- subpopulation, and the xid PP accessory cell activity was provided by an enriched population of dendritic accessory cells. These results demonstrate the the lymphoreticular cells comprising the PP microenvironment provide effective support for the differentiation of xid B cells in response to type 2 and T cell-dependent antigens.

Induction of latent immunological memory in genetically nonresponsive mice.

C57BL/10 mice exhibit major histocompatibility complex linked nonresponsiveness to hen egg white lysozyme (HEL). When these animals are primed with HEL in Freund's complete adjuvant (FCA), their secondary splenic plaque forming cell responses to aqueous HEL challenge are minimal to nonexistent. This notwithstanding, we show here that concomitant priming with both HEL and keyhole limpet hemocyanin (KLH) leads to an enhanced response to the HEL component following secondary challenge with an HEL-KLH conjugate. This enhancing effect can be transferred by nylon wool nonadherent spleen cells from HEL/FCA primed animals. Adoptive transfer studies with fractionated spleen cell populations suggest also that B cells are primed in these animals. Thus, animals which are incapable of mounting a secondary response to this antigen nevertheless appear to be primed at both the T-cell and B-cell levels following exposure to the antigen in FCA. The implications of this finding are discussed.

Immunosuppressive activities of deoxyspergualin. II. The effect on the antibody responses

We examined the suppressive effect of a newly developed antitumor agent, 15-deoxyspergualin (DSP), on the plaque-forming cell responses to various antigens. The intraperitoneal injections of DSP at a dose of 5.0 mg/kg body weight suppressed drastically the development of plaque-forming cells in the spleen of C57BL/6 mice immunized with sheep red blood cells (0.2 ml of a 5% suspension), trinitrophenyl-lipopolysaccharide (1 μg/mice) and trinitrophenyl-Ficoll(100 μg/mice). These suppressive effects were observed when the administration of DSP was started after the injection of antigens. The responsiveness of spleen cells from DSP-treated mice was also checked in in vitro culture. Responsiveness to lipopolysaccharide in vitro was not reduced. Furthermore DSP did not significantly suppress the responsiveness of spleen cells to succinylated concanavalin A or production of interleukin-1 or -2. The results are discussed with regards to the mode of action of DSP on immune responsiveness.

Cadmium-Induced Immunopathology Is Prevented by Zinc Administration in Mice

Six-week-old C57BL/6 male mice were exposed to 50 mg cadmium/L drinking water (50 ppm) for 3 wk, and killed at 0, 3 and 6 wk after cessation of treatment. In some groups, 500 mg zinc/L was added to the drinking water (500 ppm) with or after cadmium treatment. The number of direct and indirect splenic plaque-forming cells was higher in cadmium-treated mice at 0 wk than in untreated controls. Concurrent zinc administration prevented the enhancement of plaque-forming cell response. Proliferative response of spleen cell culture to the T-cell mitogens phytohemagglutinin and concanavalin A was slightly high in cadmium-treated mice at 0 wk and zinc administered after exposure to cadmium tended to lower it. The number of Lyt-2 positive cells in the spleen was lower and the ratio of L3T4 to Lyt-2 positive cells, reflecting the balance of immunoregulatory T-lymphocytes, was higher after cadmium treatment than in untreated controls. Concurrent zinc administration prevented the alteration of T-cell subsets. Cadmium and zinc treatment had no effect on liver, kidney, spleen and thymus weights and lymphocyte content of spleen, thymus and peripheral blood. Use of immunofluorescence with anti-mouse IgG and C3 showed no evidence of an autoimmune reaction in kidney sections. Liver and kidney cadmium concentrations were high at all observation times in the cadmium-treated animals. Tissue cadmium levels were lower in mice treated with both zinc and cadmium than in those treated with cadmium alone. The results suggest that a relatively low dose of cadmium exposure produces immune alterations that can be prevented by a moderately large dose of zinc.

Modulation of the immune system by Neisseria meningitidis.

The immunomodulatory potential of Neisseria meningitidis was investigated. Spleen cells from mice injected intraperitoneally with low to moderate doses of meningococci (10(4)-10(7)) were found to display enhanced responses to the mitogens lipopolysaccharide (LPS), phytohaemagglutinin (PHA), and concanavalin A (Con A). In contrast, high doses of meningococci (10(8)-10(9)) caused a marked decrease in mitogenic reactivity. Meningococci-injected mice also displayed a dose-dependent suppression of a primary anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) response. The timing between the injection of SRBC and of meningococci appeared to play an important role in the induction of suppression by the organisms. Thus, decreased PFC responses were observed only when the bacteria were injected prior to the antigen. When meningococci were injected at the same time or after SRBC, normal or even increased PFC responses developed. Kinetic experiments showed that the onset of suppression of both mitogen and antibody responses by meningococci was very rapid, so that by 6-7 h after injection of the bacteria, mice showed markedly reduced mitogen responses and became essentially unable to mount an antibody response against SRBC. Suppression of mitogen responses was relatively transient, since reactivity returned to normal after 48 h. However, the ability of infected animals to mount a normal anti-SRBC response did not fully return until 12 days after the infection. Spleen cells from meningococci-infected mice also showed markedly depressed PFC responses when stimulated with SRBC in vitro but failed to suppress the response of normal spleen cells in mixed cultures. These observations indicate that putative suppressor cells, if they exist at all, are too insignificant in terms of numbers and/or efficiency to account for the observed immunosuppression. A more likely explanation for the inhibition, which is supported by our data, presented here and elsewhere, is that certain surface components of meningococci are capable of imparting immunosuppressive signals directly onto target lymphocytes.

Early changes in T lymphocytes and subsets of mouse progeny defective as adults in controlling growth of a syngeneic tumor after in utero insult with benzo(a)pyrene

Benzo(a)pyrene, a potent carcinogen, severely suppresses the anti-SRBC plaque-forming cell response, the mixed lymphocyte response (afferent T cell function), and an in vivo graft-vs.-host response (efferent T cell function) of mouse progeny exposed to the carcinogen during gestation (11 to 13 days). Immunodeficiency occurs early after birth (1 week) and persists for 18 months. The abnormalities in the T cell-mediated responses led us to examine the quantitative profile of T cells and subsets (Lyt 1+, Lyt 2+) present in the lymphoid organs during fetogenesis (15 to 19 days) and postnatally. In addition, we examined the ability of 3- to 8-month-old progeny and their spleen cells to resist the in vivo growth of cells from a syngeneic fibrosarcoma (a tumor that had been induced by benzo(a)pyrene). Our observations included: (1) Depletion of T cells and subsets in the thymus late (19 days) in gestation and postnatally. (2) Depleted T and Lyt 1+ cells in the spleen during gestation, while postanatally the former were enhanced and the effect on the latter was variable (enhancement and reduction). (3) In the fetal liver, the T cells were reduced, but the Lyt 1+ cells were unchanged. (4) The Lyt 2+ cells were strikingly enhanced in the fetal liver and spleen, but most dramatically for the former. (5) The Lyt 1/Lyt 2 ratio was less than 1.00 or controls in the fetal liver and spleen, a condition which persisted for 30 days postnatally in the latter organ. (6) Benzo(a)pyrene-exposed progeny or their spleen cells were relatively ineffective in resisting in vivo growth of transferred tumor cells. These results show that this carcinogenic pollutant induces a marked disorientation of T cells and subsets which can persist for at least 4 weeks postnatally. This suggests disruption of T cell differentiation during ontogenesis which may have profound implications on the ability to resist induction and growth of neoplasias after in utero exposure to the carcinogen.

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation.

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.

Graft-versus-host reactions in the beige mouse. An investigation of the role of host and donor natural killer cells in the pathogenesis of graft-versus-host disease.

To investigate the role of natural killer (NK) cells in the induction and pathogenesis of graft-versus-host (GVH) disease, +/beige (+/bg; normal NK cell activity) and beige/beige (bg/bg; deficient NK cell activity) parental C57BL/6 (B6) lymphoid cells were used to induce GVH reactions in either B6 X C3H/Hej +/bg (+/bgF1) or B6 X C3H/HeJF1 bg/bg (bg/bg F1) hybrid mice. When B6 bg/bg parental lymphoid cells (PLC) were injected into bg/bg F1 mice, early splenomegaly, early severe suppression of the plaque-forming cell (PFC) response to sheep red blood cell (SRBC), and only partial suppression of T cell mitogen responses to concanavalin A (Con A) and phytohemagglutin (PHA) were observed on day 12 after GVH induction. In the same GVH combination, slightly augmented NK cytotoxic activity was induced and no GVH-induced moderate-to-severe pathological alterations in the liver and pancreas were observed. When bg/bg PLC were injected into +/bg F1 mice, early splenomegaly and pronounced immunosuppression of the PFC response to SRBC and partial suppression of Con A and PHA responses were observed on day 12 after GVH induction. In this combination (bg/bg----+/bg F1), significant NK cell activity was induced, but no moderate-to-severe histopathological alterations were observed. In contrast, when B6 +/bg PLC were injected into either +/bg F1 or bg/bg F1 hybrids, early splenomegaly, and severe immunosuppression of both the PFC response to SRBC and the T cell mitogen responses to Con A and PHA were observed by day 12--which persisted until day 30 after GVH induction. Furthermore, high NK cell activity was recorded and moderate-to-severe histopathological alterations appeared in both +/bg F1 and bg/bg F1 recipients. These results show that the bg/bg PLC can induce GVH-associated early splenomegaly and immunosuppression of the PFC response to SRBC in both the bg/bg F1 and +/bg F1 hybrids, but that it failed to induce moderate-to-severe histopathological alterations, even though NK cell activity of host origin was activated during GVH reactions. Conversely, when +/bg donor cells were used to induce the GVH reaction, splenomegaly and immunosuppression, as well as moderate-to-severe histopathological lesions were induced. These results suggest that donor NK cells rather than host NK cells play an active role in GVH-associated tissue damage, which in turn contributes to the long-term suppression.

KINETICS OF NATURAL KILLER CELLCYTOTOXICITY REACTION

The relationships between splenic natural killer (NK) cell cytotoxicity, T and B cell function, and the development of histopathological lesions in the liver and pancreas have been studied during the course of graft-versus-host (GVH) reactions. GVH reactions were induced in (C57BL/6A)F1 (B6AF1) hybrids by different doses, (10,20 and 30×106) of either parental strain C57/BL6 (B6) or A lymphoid cells. Splenic NK cell cytotoxicity was studied by employing YAC-1, an NK-eellsensitive target. Splenic T and B cell function were assessed by mitogen responsiveness to concanavalin A, phytohemagglutiain, and Escherichia coli lipopolysaccharide, and by the in vitro plaque-forming cell response to sheep red blood cells. Histopathological lesions characteristic of GVH reactions were recognized at a time (day 8 after GVH induction) when both T and B cell functions were totally suppressed and NK cell activity was greatest. The severity of histopathological alterations later (day 16 after GVH induction) correlated with an early peak in NK cell cytotoxicity rather than with the overall NK cell activity. When low doses (10,20×106) of B6 cells were employed to induce GVH reactions, a significant increase in NK cell activity was observed, yet neither histopathological alterations nor suppression of T and B cell functions were observed. The killing of YAC-1 targets by splenocytes obtained from the different GVH combinations could not be abrogated by pretreatment with anti-Thy 1.2 serum plus complement, suggesting that T lymphocytes were not central to this cytolytic process. These experiments demonstrated that: (1) an inverse relationship between T and B cell function and NK cell activity was observed early after GVH induction, (2) the severity of histopathological lesions and immunosuppression, as well as the degree of overall augmented NK cell activity, was determined by the dose and genotype of donor cells injected to induce GVH reactions, and (3) GVH-associated moderate-severe lesions occurred only in groups in which NK cell activity peaked early—-whereas-when NK cell activity peaked later, either mild or no lesions were observed, suggesting that the early rapid increase of NK cell activity may be useful for predicting the severity of GVH pathogenesis.