Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide, and other bacterial products.

The growth of WEHI-231, a murine immature B lymphoma cell line, was inhibited by anti-IgM antibodies. The inhibition of proliferation, as measured by [3H]thymidine incorporation, occurred between 16 and 28 hr after addition of anti-IgM. Moreover, the growth arrest was irreversible: cells that were cultured with anti-IgM for 18 hr and then recultured without it failed to recover the ability to proliferate, even though cells treated for up to 30 hr with anti-IgM remained viable, as measured by trypan blue exclusion. Three polyclonal B cell activators obtained from bacteria, lipopolysaccharide (LPS), peptidoglycan from Staphylococcus aureus, and gliding bacterial adjuvant from Cytophaga (GBA), were able to protect WEHI-231 cells from anti-IgM-induced growth arrest. The protection was transient, ending after approximately 56 hr. This transience was shown to be due to desensitization of the cells to the bacterial products. Interestingly, pretreatment of WEHI-231 cells with any of the bacterial products desensitized the cells to all of the bacterial products. The heterologous nature of this desensitization suggests that all three of these bacterial products may act through a common signaling pathway despite their diverse chemical natures.

Soluble non-cross-linked peptidoglycan polymers stimulate monocyte-macrophage inflammatory functions.

Soluble non-cross-linked peptidoglycan polymers are released by gram-positive bacteria when beta-lactam antibiotics are administered to humans. In this report, we show that this type of peptidoglycan can stimulate monocyte-macrophage functions that cause inflammation. Non-cross-linked peptidoglycan polymers from penicillin-treated Streptococcus faecium were purified and shown to stimulate the production of interleukin 1 by human monocytes and of colony-stimulating factors by a murine macrophage cell line. In addition, the release of plasminogen activator by human monocytes was inhibited by the soluble peptidoglycan. These in vitro results suggest that prolonged treatment with beta-lactam antibiotics, by causing the production of soluble peptidoglycan, may result in interleukin 1-mediated inflammatory reactions, excessive production of monocytes and granulocytes, and increased fibrin deposition.

T cell-derived glucosteroid response-modifying factor (GRMFT): a unique lymphokine made by normal T lymphocytes and a T cell hybridoma.

Mouse spleen cells and a murine T cell hybridoma, FS6 14.13.1, produce a glucosteroid response-modifying factor (GRMFT) after stimulation with concanavalin A. GRMFT blocks glucosteroid suppression of helper T cell function and the growth of granulocyte/macrophage progenitor cells in vitro. IL 1 also protects helper T cells and myeloid precursors from glucosteroid suppression. This suggests that GRMFT and IL 1 act congruently to ensure that an effective immune response is generated when endogenous glucosteroid levels are elevated. To understand the role of GRMFT in normal immune responses and in disease states characterized by imbalances in the immune system, we began to purify and characterize GRMFT. GRMFT appears to be distinct from other well-characterized T cell-derived factors. GRMFT is larger than IL 2 as determined by gel exclusion chromatography and is completely separated from IL 2 by isoelectric focusing. Furthermore, purified IL 2 does not have GRMFT activity. Purified IL 3 also lacks GRMFT activity, and conditions that inactivate immune interferon have no effect on GRMFT. Thus, GRMFT is different from IL 2, IL 3, and immune interferon. GRMFT also lacks activity in the thymocyte co-mitogenic assay and is therefore different from IL 1. Finally, FS6 14.13.1 reportedly does not produce TRF or CSF, which suggests that GRMFT is different from these molecules as well.

Prevention of the in vitro myelosuppressive effects of glucocorticosteroids by interleukin 1 (IL 1).

We investigated the effects of dexamethasone on the formation of granulocyte/macrophage colonies by murine bone marrow cells cultured with colony-stimulatory factors (CSF) in semisolid agar. Dexamethasone (10(-7) M) completely inhibited the formation of colonies in response to L929 CSF but had no effect on the response to CSF in the culture supernatants of the murine macrophage cell line, PU5-1.8. We postulated that a cofactor, interleukin 1, present in the PU5-1.8 supernatants was responsible for protecting colony formation against steroid suppression. Interleukin 1, isolated from culture supernatants of PU5-1.8 and from culture supernatants of human acute monocytic leukemia cells, blocked the inhabitory effects of dexamethasone on colony formation in response to L929 CSF. Moreover, dexamethasone inhibited colony formation in response to PU5-1.8 culture supernatants when interleukin 1 was absent. We also examined interleukin 2 for possible protective effects. Although crude interleukin 2 preparations (supernatants of spleen cells cultured with concanavalin A) blocked dexamethasone inhibition, purified interleukin 2 had no protective effects. These data indicate that interleukin 1 protects colony formation by a pathway that is independent of interleukin 2 and that supernatants of spleen cells activated with concanavalin A probably contain significant amount of interleukin 1.

Differential effects of glucocorticosteroids on the functions of subpopulations of helper T lymphocytes.

The in vitro effects of dexamethasone on the activities of Th1 and Th2 helper cell subpopulations were examined in secondary (IgG) responses to hapten-carrier conjugates under conditions where the selective or dominant expression of their individual activities could be observed. In the induction of IgG anti-hapten responses, carrier-primed Th1 cells cooperate with hapten-primed B cells by linked recognition of antigen, while carrier-primed Th2 cells cooperate with hapten-specific B cells by unlinked recognition of antigen. The function of carrier-primed Th1 cells was resistant to inactivation by dexamethasone. In contrast, the function of carrier-primed Th2 cells was abolished in the presence of pharmacologic concentrations of the steroid. The differential effects of dexamethosone on the activities of the two subpopulations of helper T cells could not be attributed to selective inhibition of a subpopulation of B cells which cooperate with Th2 cells.

Differential effects of glucocorticosteroids on the functions of helper and suppressor T lymphocytes.

The effects of dexamethasone (Dex) on the functions of antigen-primed helper and suppressor T cells were studied in humoral immune responses in vitro. In doses equivalent to elevated physiologic concentrations, the suppressor T cell activity was abolished. In contrast, the helper T cell function was resistant to even pharmacologic concentrations of Dex. The apparent steroid resistance of the helper T cells was found to be mediated by the products of activated macrophages. While macrophage factors protected helper T cells from steroid inhibition, they did not prevent the effects of Dex on suppressor T cells. Because bacterial cell wall and membrane components are potent inducers of the factors that mediate steroid resistance of helper T cells, the combination of physiologically elevated levels of steroids and macrophage factors during acute infections may function to facilitate the expression of host immunity. However, the persistance of these conditions, as in chronic inflammation, may also contribute to the pathogenesis of autoimmunity by perturbing the balance of immune regulation by helper and suppressor T cells.

Prevention of the immunosuppressive effects of glucocorticosteroids by cell-free factors from adjuvant-activated accessory cells.

Cultured mouse spleen cells are protected from the immunosuppressive effects of glucocorticosteroid hormones by prior treatment with bacterial adjuvants. The mechanisms by which such protection is acquired were further invgestigated in this study. Cell-free factors, termed glucocorticosteroid response modifying factors (GRMF), were obtained from the supernatant fluid of normal resident murine peritoneal macrophages activated in vitro by treatment with lipopolysaccharide. These factors provided protection against steroid immunosuppression in a dose dependent fashion. Two-step experiments show that GRMF treatment results in the recovery of significant amounts of T helper activity which otherwise is suppressed by steroids. Factors with similar biologic activities were also obtained from murine monocytic cell lines. The possible mechanisms by which GRMF protects the immune response are discussed.

Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens.

Immunodepression in Taenia crassiceps infection: restoration of the in vitro response to sheep erythrocytes by activated peritoneal cells.

The in vitro response to sheep erythrocytes of mesenteric lymph node cells from mice infected with the larval cestode Taenia crassiceps is significantly depressed and can be restored to control levels by addition of activated peritoneal cells depleted of functional T or B lymphocytes. Adherent mesenteric lymph node cells from infected mice are unable to reconstitute the in vitro response to sheep erythrocytes of normal nonadherent cells. The responses of mesenteric lymph node cells from infected mice to the T-lymphocyte mitogens concanavalin A and phytohemagglutinin and the B-lymphocyte mitogen lipopolysaccharide are normal. Mesenteric lymph node cells from infected mice do not suppress the in vitro response to sheep erythrocytes of normal mesenteric lymph node cells. These results suggest that the immunodepression in T. crassiceps-infected mice is primarily the result of alterations in functional accessory cells.

Black under-representation in United States medical Schools.

To understand why blacks are under-represented in medical schools and how this situation might be changed, we analyzed statistics on medical-school applicants, medical-school students and college undergraduates. The pool of qualified black applicants to medical schools has not been large enough to achieve appropriate black representation. If black under-representation is to be corrected, the pool of qualified black applicants must be increased. Affirmative-action programs for blacks who are already in college are unlikely to be sufficient by themselves to increase the pool of black applicants substantially. Such programs should therefore be developed for high-school students.

Sera and the in vitro induction of immune responses. III. Adjuvant obtained from gliding bacteria with properties distinct from enteric bacterial lipopolysaccharide.

We have extended our studies on the role of bacterial contamination in the generation of fetal calf sera which support primary in vitro humoral responses by cultured mouse spleen cells. Gram-negative, gliding bacteria were isolated from a strongly supportive sample of fetal calf sera. Medium conditioned by the growth of these microorganisms had strong adjuvant effects in cultures of mouse spleen cells supplemented with a non supportive, deficient sera. The adjuvant and mitogenic activities of the bacterial conditioned medium were then compared to those of bacterial lipopolysaccharide from Salmonella typhosa 0901 in cultures of LPS responder and LPS nonresponder spleen cells. From the results of these comparative studies, we conclude that the active factor(s) obtained from the gliding bacteria is an adjuvant with properties very distinct from those of highly purified enteric bacterial LPS.

The role of activated accessory cells in preventing immunosuppression by hydrocortisone.

The generation of humoral immunity in vitro by normal and antigen-primed mouse spleen cells was suppressed by in vitro treatment with hydrocortisone. Functions of normal and antigen-activated helper T lymphocytes and of accessory cells were inhibited by the corticosteroids. Spleen cells cultured overnight in medium containing fetal bovine serum became highly resistant to the effects of hydrocortisone. Similar resistance was found to occur when spleen cells were cultured with accessory cells that previously had been activated with bacterial lipopolysaccharide. These studies show that immunologically nonspecific processes significantly alter the effects of the steroids on specific immune responses and suggest that accessory cell products modulate T cells in ways which differ from antigen induction.

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.

Functional deficiency of splenic adherent cells in New Zealand black mice.

New Zealand Black (NZB) mice hyporespond after in vitro immunization to sheep erythrocytes but not after in vivo immunization. The difference between in vitro and in vivo immunization was found to involve a non-antigen-specific change in the spleen which occurs in vivo soon after priming. Adherent spleen cells from young NZB or control strain mice were compared in their ability to cooperate with nonadherent cells in the induction of a primary immune response to sheep erythrocytes in vitro. The results support the hypothesis that there is a functional deficiency of adherent cells in the spleens of unprimed NZB mice.

Sera and the in vitro induction of immune responses. I. Bacterial contamination and the generation of good fetal bovine sera.

More than 200 samples of pooled fetal bovine sera (FBS) were tested to determine their capacity to support primary immune responses by cultured mouse spleen cells. Less than 10% of the FBS samples were fully supportive; the majority of the samples were moderately to very deficient. Several lines of evidence suggest that bacterial contamination during the processing of sera plays a major role in determining which sera are supportive. Serum samples known to have been temporarily contamined during processing were strongly supportive. Samples of FBS which were likely never to have been contaminated were deficient. Bacteria from a specific lot of supportive serum converted a very deficient serum into one which was supportive. Several mechanicsms by which bacteria could effect sera are discussed.