Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.     

Suppressor cell influence in selected strains of inbred rats: I. Strain-dependent mitogen- and antigen-related low responsiveness

Lymphocytes derived from Lewis (LE), Brown-Norway (BN), or the F₁ hybrid (LBNF₁) rats respond in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. The magnitude of the response, as determined by incorporation of ³H-thymidine, ¹⁴C-adenine, and ³H-leucine, was highest for LE and lowest for BN animals. These proliferation response differences were observed for lymph node lymphocytes and peripheral blood lymphocytes. The response to antigen, as measured by lymphocyte transformation, reflected the mitogen responsiveness of the strains tested, i.e., LE animals responded to a higher level than did BN animals. Equivalent levels of antibody were found in all animals immunized with antigen. In addition, BN rats are suppressed to a greater magnitude than are LE rats when both strains are primed, rechallenged, and assayed via lymphocyte transformation to the test antigen.     

Suppressor cell infleunce in selected strains of inbred rats. II. Macrophage-associated suppression of cell-mediated immune responsiveness.

Responses to concanavalin A or antigen by allogeneic combinations of Lewis (LE) and Brown-Norway (BN) rat derived lymphocytes and macrophages were of comparable magnitude to responses by the syngeneic combinations. But at increased concentrations of macrophages to lymphocytes, suppression of lymphocyte reactivity, as assayed by incorporation of tritiated thymidine, was evident. Suppression associated with BN derived macrophages alone, or in combination with LE or Buffalo derived macrophages, was consistently of a greater magnitude. Possible explanations of the macrophage associated suppression are discussed.     

Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Nitric oxide-induced anti-mitogenic effects in high and low responder rat strains.

Nitric oxide (NO) has multiple biologic functions: in the brain it acts as a neuronal messenger; elsewhere, it causes smooth muscle relaxation, inhibition of platelet aggregation, inhibition of leukocyte adhesion, inhibition of tumor growth, and microbiostasis. Our studies show that production of NO is responsible for the unusual unresponsiveness of BN rat spleen cells to mitogens. NG-monomethyl-L-arginine (NGMMA), a potent competitive inhibitor for NO synthase, reverses this defect. Lysed RBC or NGMMA were shown to enhance mitogen-induced spleen cell proliferation only one- to twofold in Lewis rats (that have normal mitogen responsiveness) but act to stimulate BN rat T cells by 10- to 100-fold. NGMMA-enhanced proliferation was significantly diminished by prior depletion of macrophages. Surprisingly, NO did not inhibit IL-2 production in 48-h cultures of BN rat spleen cells, and exogenous IL-2 was ineffective in releasing NO-mediated suppression. These studies indicate that NO produced by macrophages can completely and reversibly inhibit T cell proliferation. The BN rat appears to be unique in its production of very high levels of NO, making it an especially useful animal model for studying the biologic control and functional consequences of NO generation.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Accessory cell function of thoracic duct nonlymphoid cells, dendritic cells, and splenic adherent cells in the Brown-Norway rat

Thoracic duct lymph of lymphadenectomized Brown-Norway (BN) rats is highly enriched for nonlymphoid cells (NLC) which share several characteristics with splenic dendritic cells (DC), e.g., the binding of monoclonal antibody OX2. The accessory cell activity of NLC was analyzed by comparing these cells with DC and splenic adherent cells (SAC). In concanavalin A (Con A)-induced T-cell proliferation NLC, like DC, were very effective accessory cells at low cell numbers, as a consequence of an efficient induction of interleukin 2 (IL-2) production and IL-2 responsiveness. Responses in the presence of SAC were poor, even after the addition of excess IL-2. A fourfold enhancement of accessory cell activity of SAC was achieved by the depletion of FcR-positive cells, which were responsible for suppression of the Con A response. Low responsiveness of BN rats with respect to Lewis rats can in part be explained by a higher suppressive activity of macrophages in the BN rat.     

Role of non-RT1 genes in the response of rat lymphocytes to Mycoplasma arthritidis T cell mitogen, concanavalin A and phytohemagglutinin

A comparison of splenic cells from various inbred rat strains indicated that DA, Lewis, Buffalo, August, Wistar Furth, and (LEW X BN)F1 all responded well to the Mycoplasma arthritidis T cell mitogen, phytohemagglutinin and concanavalin A, but cells from BN and MAXX rats were very weakly or nonresponsive. Cells from congenic strains expressing nonresponder background genes, and responder haplotypes at RT1 (BN.1L(LEW), RT1; BN.1A(DA), RT1av1) failed to respond significantly to the mitogens. Rats expressing responder background genes but the nonresponder haplotype at RT1 at RT1 (WF.1N-(MAXX), RT1n) exhibited high responses to all mitogens. The controlling role of non-RT1 genes was confirmed by testing tissue-typed (DA X BN)F2 progeny and (DA X BN)F1 X DA and (DA X BN)F1 X BN progeny. No association was seen between the expression of a/a, a/n, or n/n at RT1 and the degree of response to the mitogens. In contrast, as the proportion of DA non-RT1 genes increased, so did the degree of mitogenic responsiveness. Similar results were obtained by using a partially purified preparation of the mycoplasma T cell mitogen. The results indicated that in the (DA X BN)F1 hybrids, responsiveness to all mitogens was recessive: this contrasts with the (LEW X BN)F1 hybrids in which responsiveness was dominant. Finally, we showed that both responder and nonresponder splenic cells were capable of binding the M. arthritidis mitogen. The data contrast with those obtained with nonresponder mouse strains the cells of which failed to bind mitogen due to the absence of the E alpha chain of the I-E-coded molecule.     

Comparison of the tube leukocyte adherence inhibition test with the lymphocyte stimulation assay in a rat transplantation model.

The tube leukocyte adherence inhibition test (LAI) was analyzed in a heterotopic heart transplantation model in BN and Wag/Rij rats. Seven, fourteen, and twenty-two days after transplantation spleen cells of the recipient rat were tested for recognition of histocompatibility antigens in crude membrane extracts prepared from normal rat tissue. It was found that addition of fetal calf serum affected the cell adherence considerably and that there was a large difference in the percentage of adherent cells from different animals. In a comparative study the lymphocyte stimulation assay proved to be more sensitive and reproducible than the LAI in this rat system.     

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. I. Generation of suppressor macrophages in spleen cells of BCG-vaccinated mice.

With the aim of clarifying the mechanism of the suppressive action of BCG against insulitis and overt diabetes in NOD mice, we studied the effects of BCG on spleen cell populations and on the in vitro immune responses of spleen cells. The spleen cells of BCG-vaccinated mice showed much lower responsiveness to various mitogens such as Con A, PHA, PWM, and LPS than those of saline-treated mice. Low responsiveness to alloantigens was also observed. Flow cytometric analysis of the spleen cells revealed that Mac-1+ and Mac-2+ cells had increased while T and B cells had decreased in the BCG-vaccinated mice compared with the saline-treated mice at the time when the maximum level of inhibition of mitogen responses of BCG-vaccinated mice was observed. This suggests that the decreased in vitro immune response was due to the increase in macrophages which suppress lymphocyte functions. Support for this interpretation comes from the following two findings: (1) the restoration of mitogen responses of spleen cells when macrophages were eliminated by plastic adhesion or FACS sorting and (2) resuppression of PHA and Con A responses of plastic-nonadherent spleen cells by addition of adherent cells or flow cytometrically sorted Mac-1+ cells obtained from BCG-vaccinated mice. These results indicate the generation of suppressor macrophages after BCG vaccination and suggest that these macrophages prevent the autoimmune pathogenesis leading to diabetes in NOD mice.     

Heterogeneity of suppressors of mitogen responsiveness in human malignancy

Summary Blood lymphocytes from cancer patients frequently showed reduced responsiveness to mitogens compared with those from healthy individuals. This impairment did not appear to be attributable to an intrinsic T cell deficit but to the involvement of suppressor cells. The role of monocytes as suppressors in cancer patients was supported by experiments in which they were depleted by removal of adherent cells or selectively eliminated in situ by treatment with macrophage toxic agents (carrageenan, silica). Phytomitogen responses were consistently elevated by these manoeuvres. Indomethacin elevated responses in unseparated but not adherent cell depleted populations, indicating an important role for prostaglandins in this phenomenon. None of these procedures markedly influenced the response of lymphocytes from healthy individuals.Following removal of adherents, response could be generated or increased in SRBC rosetting or non-rosetting fractions depending upon the initial responsiveness of the adherent depleted populations, an observation consistent with the coexistence of responder and suppressor populations. Tumour-draining lymph node cells differed from peripheral blood insofar as no evidence of monocyte-like suppressors was found, but SRBC rosetting cells depressed the mitogen stimulation of autologous blood lymphocytes.These data establish that suppressor cells are operative in the depression of T cell responses to mitogens and indicate that several cell types are involved.     

In vitro anamnestic antibody response: differential cellular and calcium requirements for induction by antigen and regulation by cyclic AMP.

Lymph node cells from rabbits, immunized 6 clays previously with keyhole limpet hemocyanin (KLH) were fractionated on columns containing nylon fibers. The non-retained population (effluent cells) and the retained population (adherent cells) were subsequently characterized by various criteria. The addition of dibutyryl cAMP (DbcAMP) or cholera enterotoxin (CT) during induction by 1 and 100 μg KLH resulted in a >100% increase in antibody synthesis over the control (KLH only) responses in the unfractionated and adherent cell populations. In the effluent population CT and DbcAMP failed to enhance the 1 μg response, but did increase the 100 μg response. Antibody forming cells, as judged by ongoing antibody synthesis during the first 24 hr of culture, were deficient in the effluent population. Both the effluent and adherent cells responded to the mitogens concanavalin A, phytohemagglutinin, and goat anti-rabbit Fab'. The control, effluent, and adherent populations each contained approximately 45% surface Ig positive cells as judged by direct immunofluorescence. The removal of calcium from the medium during induction (0–24 hr) also demonstrated that induction of the antibody response by KLH was separable from the cAMP mediated enhancement of antibody synthesis.     

Immunologic deficiency during experimental Chagas' disease (Trypanosoma cruzi infection): role of adherent, nonspecific esterase-positive splenic cells

The involvement of adherent splenic cells in the production of deficient lymphocyte responses during the acute phase of experimental Chagas' disease was investigated. When cultured together, purified adherent splenocytes from mice acutely infected with Trypanosoma cruzi caused a significant reduction in the responses of normal mouse spleen cells to T and B cell-specific mitogens. Similar observations were made when infected mouse adherent splenocytes were co-cultured with normal mouse nonadherent cells. Exchange of adherent cells in infected mouse spleen cells suspensions for adherent cells from uninfected mice resulted in increased responses to stimulation with the T and B cell mitogens tested. Treatment of infected mouse cell suspensions with indomethacin improved the responsiveness of these cells to the mitogens. These results support the concept that the immunosuppression that is characteristic of experimental acute Chagas' disease is at least in part mediated by an adherent cell population and is dependent on a prostaglandin-mediated mechanism.     

In vitro inhibition of lymphoproliferative responses to tumor associated antigens and of lymphoma cell proliferation by rat splenic macrophages.

Spleens from W/Fu rats bearing a syngeneic progressively growing (C58NT)D tumor contain cells which can inhibit lymphoproliferative responses in a mixed lymphocyte-tumor interaction designed to demonstrate suppressor activity. Spleens from rats having rejected (C58NT)D tumors also contained suppressor cells but to a lesser degree. The growth inhibition assay, which measures inhibition of proliferation of tumor cells, was evaluated as a simple assay system to screen for suppressor cell activity. The effector cells in both assays had the same characteristics, indicating a predominant role of macrophages. Normal rat spleens were found to contain growth inhibition activity which led to the demonstration of suppressor cell activity in spleens of normal animals. Removal of suppressor cells from the spleens of immunne rats results in consistently higher lymphoproliferative responses to tumor associated antigens on the tumor cells.     

A suppressor cell which interferes with antiallotype stimulated DNA synthesis of rabbit B cells

Responsiveness of rabbit spleen cells to anti-allotype antibody was measured in terms of increased thymidine incorporation. Incorporation was enhanced after removal of cells which had ingested or had adhered to magnetic particles. B lymphocytes, prepared from spleen cells by the removal of adherent cells and of RTLA bearing T cells, were more responsive to anti-allotype antibody than were the original spleen suspensions. This increase could not be explained by enrichment in B cells. It was concluded that an adherent cell suppressed B cell transformation. The addition of 2-mercaptoethanol to the cell cultures stimulated with mitogen augmented the incorporation of thymidine. Adherent cells interfered with 2-mercaptoethanol potentiation in the response to anti-allotype antibody but not in the response to Con A. Fractionation of spleen cells, over glass bead columns, yielded nonadherent and adherent cell populations. The responsiveness of nonadherent cells to anti-allotype induced thymidine incorporation was two to six times that of unfractionated cells. The responsiveness of nonadherent cells to stimulation by anti-allotype antibody was reduced after addition of adherent cells. Findings were discussed in terms of the inhibitory role played by adherent cells on anti-allotype antibody induced responsiveness of rabbit B cells and of the possible participation of a third cell type which functions as a promotor of mitogenic T cell stimulation.     

Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage activation during experimental murine brucellosis: II. Inhibition of in vitro lymphocyte proliferation by brucella-activated macrophages

During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

Sex-related immunocompetence of BALB/c mice. I. Study of immunologic responsiveness of neonatal, weanling, and young adult mice.

The responses of lymphoid cells from the thymus, lymph nodes, and spleen of male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. Immunologic assays used included blastogenic responses to mitogens, mixed lymphocyte responses, and direct and indirect measurement of plaque-forming cells against soluble and particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Little or no differences between the sexes were observed in the mitogenesis of lymph nodes and thymuses. Newborn mice did not demonstrate the sex-associated immune differences. Among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed.     

Inhibition of lymphocyte blastogenesis by C3c and C3d.

The C3 cleavage products C3c and C3d were tested for their ability to alter the immunoproliferative response of human peripheral mononuclear cells to the antigens SLO and SK-SD, and to the mitogens PHA and PWM. It was found that both C3c (30 to 120 micrograms/ml) and C3d (10 to 40 micrograms/ml) inhibited lymphocyte blastogenesis in the presence on antigens but not mitogens, when cells were cultured in either autologous plasma or FCS. Similarly, the response to antigens of cell populations enriched for T lymphocytes was inhibited, whereas the response to optimal or suboptimal doses of mitogens was unaffected. When nonadherent (NA) cells were reconstituted with increasing numbers of adherent (AD) cells to potentiate the proliferative response of NA cells to the antigen SLO, the addition of either C3c or C3d abolished the potentiation of the response at low levels of reconstitution. However, at given dose of C3c or C3d, addition of excess AD cells could restore the proliferative response. These results suggest that both C3c and C3d can inhibit T cell proliferation in response to antigen and that they may act at the level of the monocyte-T lymphocyte interaction to modulate cellular immune responses.