Inability of mitogen-activated lymphocytes obtained from patients with malignant primary intracranial tumors to express high affinity interleukin 2 receptors.

Patients with primary malignant brain tumors manifest a variety of abnormalities in cell-mediated and humoral immunity. Diminished T cell reactivity has been shown in these patients to be linked to deficiencies in interleukin 2 (IL-2) production that cannot be overcome by exogenous IL-2. In this study, specific binding of radiolabeled IL-2 to PHA-stimulated lymphocytes from brain tumor patients demonstrates that the number of high affinity interleukin 2 receptors (IL-2R) is greatly reduced. FACS analysis indicates that the relative density of the p55 protein (Tac protein) is lower on the mitogen-activated lymphocytes obtained from patients than on comparably treated lymphocytes from normal individuals. These data indicate that mitogen-stimulated lymphocytes obtained from patients have fewer functional high affinity IL-2R principally because of the failure to express sufficient levels of the p55 protein for association with the p75 protein. Northern analysis of total RNA isolated from mitogen-stimulated T cells from patients demonstrates normal levels of steady state mRNA, which codes for the p55 protein. Moreover, there is no defect in the postranslational processing of the primary translation product of this mRNA suggesting that normal levels of the p55 protein are produced in activated T cells from patients.

General immunocompetence of rats bearing avian sarcoma virus-induced intracranial tumors.

The mitogenic responsiveness of spleen cells obtained from avian sarcoma virus-inoculated Fischer 344 rats was studied. Sixty % of the rats had astrocytomas, 13% had sarcomas, 7% had mixed gliosarcomas, and 20% had no evidence of tumors. Only spleen cells from rats bearing astrocytomas had significantly diminished responses to phytohemagglutinin and concanavalin A (Con A) when compared to control responses. The decreased responsiveness observed with phytohemagglutinin was limited to the optimal concentration range (10 and 20 microgram) while a broader concentration of Con A (0.01 to 50 microgram) induced significant suppression. Moreover, a more profound immunosuppression was observed with Con A. The results also demonstrated that spleen cells from rats with the largest astrocytomas exhibited the greatest suppression. From the results of this study, it appears the avian sarcoma virus-induced astrocytoma in rats is an immunological parallel of the human disease based on the loss of general immunological competence as assessed by responsiveness of lymphocytes to phytohemagglutinin and Con A.

Neuromodulation of lymphocyte reactivity in aged rats.

Numerous reports indicate that both central nervous system (CNS) and immune system functions decline with age. We have previously shown that the CNS can modulate both mitogen-induced spleen cell proliferation and NK activity in young Fischer 344 rats. In the present study we have determined the effects of AHT lesions on the lymphocyte reactivity of aged Fischer 344 rats. These data show that lesions in the AHT of aged rats cannot modulate splenic mitogen responsiveness, however, NK activity is impaired. This differential effect may be due to multiple factors including enhanced splenic suppressor cell activity, the inability of the brain to send modulatory signals following lesioning, or the failure of the immune system to receive a neural signal and react to it.

Role of spontaneous interstitial cell testicular tumors on the mitogen reactivity of spleen cells from aged male Fischer-344 rats.

Virtually all aged, male, Fischer-344 rats have testicular tumors. The influence of this tumor on lymphocyte reactivity from aged Fischer-344 rats is unknown. In this report we demonstrate that neither the presence of this tumor nor the serum concentration of luteinizing hormone has an effect on the splenic mitogen reactivity of old animals.

Transplantation of pituitary grafts fail to restore immune function and to reconstitute the thymus glands of aged mice.

There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.

A heat-stable blocking factor in the plasma of patients with subacute sclerosing panencephalitis.

The possible role of a defective cell-mediated immune response in the pathogenesis of subacute sclerosing panencephalitis remains unclear. Cell-mediated immunity has been investigated in nine patients with this disorder. In eight patients, a heat-stable blocking factor in the plasma inhibited normal lymphocyte transformation to phytohemagglutinin and mixed lymphocyte cultures. The degree of blocking increased as the disease progressed. The nature of the blocking factor is unknown and is currently under investigation. A heat-stable blocking factor has not been described previously in subacute sclerosing panencephalitis, althouth a heat-labile blocking factor has been reported in a total of five patients.

Effects of preincubating human peripheral blood lymphocytes on their ability to bind sheep red blood cells.

The effect of preincubating human peripheral lymphocytes at 37 degrees C or 4 degrees C various lengths of time on their subsequent ability to form rosettes with sheep red blood cells (SRBC) was investigated. Lymphocytes suspended in either medium or medium containing fetal calf serum (FCS) and preincubated at 37 degrees C for 30 min exhibited marked decrease in rosette formation with a return to normal values by 120 min. However, neither lymphocytes suspended in medium and preincubated at 4 degrees C nor lymphocyte suspended in medium containing normal human serum (HS) and preincubated at 37 degrees C exhibited this phenomenon.

Neural modulation of immune function.

In this report we review our hypotheses and approaches to the study of the relationship between the central nervous and immune systems. Discussed are results pertaining to the modulation of immune parameters resulting from perturbations of the brain employing electrolytic lesions and the neuroleptic 6-hydroxydopamine. Experiments describing the central and peripheral effects of serotonin on in vivo and in vitro immune responses are also discussed.

Central catecholamine depletion impairs in vivo immunity but not in vitro lymphocyte activation.

We have previously shown that depletion of central nervous system (CNS) catecholamines by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the cisterna magna of C57B1/6 mice markedly impairs the humoral immune response to sheep red blood cells. This work extends these observations by showing that 6-OHDA treatment also inhibits the humoral antibody response to the T-cell-dependent antigen trinitrophenyl-keyhole limpet hemocyanin, but does not affect the response to the T-independent antigen trinitrophenyl-lipopolysaccharide. This treatment also impairs humoral responsiveness at peripheral lymphoid sites in addition to inhibiting natural killer cell activity. However, 6-OHDA treatment in vivo does not affect in vitro mixed lymphocyte responsiveness, mitogen-induced lymphocyte activation or antigen presentation by macrophages.

Potentiation of antibody responsiveness after the transplantation of a syngeneic pituitary gland.

Transplantation of a pituitary graft under the kidney capsule and the resulting elevation of serum prolactin enhances the primary humoral antibody response to sheep red blood cells. Enhancement of the response is not due to marked changes in the percentage of T-cells and their subsets, B-cells, or the number of nucleated spleen cells. Quantitation of serum prolactin levels correlates well with the proportion of enhancement as mice with two grafts and higher levels of prolactin have increased responsiveness compared to mice with one graft. Systemic administration of mouse prolactin at the time of immunization also enhances the humoral immune response; however, if prolactin treatment is delayed and given 24 h after immunization, no potentiation of the response occurs. Thus, prolactin is enhancing the immune response by affecting an early afferent event in the induction of the immune response.

Neurotransmitter-lymphocyte interactions: dual receptor modulation of lymphocyte proliferation and cAMP production.

Stimulation of the beta-adrenergic receptor on lymphocytes can decrease the proliferative response of these cells to mitogens. We have found that simultaneous stimulation of T cells with the beta-adrenergic agonist isoproterenol and mitogens (phytohemagglutinin (PHA) and OKT3 monoclonal antibody) results in a 2- to 4-fold increase in cAMP production compared to cells exposed to isoproterenol alone. Mitogens alone have little effect on cAMP synthesis, but do activate the phosphatidylinositol (PI) cycle, suggesting that interactions may be occurring between the second messenger systems resulting in a cAMP synergy. Further experiments suggest that calcium may be involved in inducing the cAMP synergy observed in T cells. It is proposed that the synergy between beta-adrenergic and mitogenic stimulation of T cells for cAMP may be involved in the mechanism of catecholamine modulation of lymphocyte function.

cAMP accumulation in T-cells inhibits anti-CD3 monoclonal antibody-induced actin polymerization.

The results presented in this report offer a novel explanation for how stimulation of the beta-adrenergic receptor (beta AR) inhibits the ability of T cells to proliferate after interaction with immobilized anti-CD3 monoclonal antibody (mAb). Accordingly, T cells binding to immobilized anti-CD3 mAb but not anti-CD4 mAb undergo time-dependent F-actin assembly with concomitant formation of pseudopodia. This process is completely inhibited in the presence of isoproterenol (ISO) indicating that stimulation of the beta AR on T cells interferes with the biochemical processes responsible for the assembly of actin. To confirm these observations, we quantitated the formation of F-actin in T cells stimulated with immobilized anti-CD3 mAb in the presence of cAMP elevating agents. The results show that stimulation of the beta AR on T-cells, as well as the addition of forskolin or dibutyryl cAMP, abrogates the formation of F-actin.

Immune defects observed in patients with primary malignant brain tumors.

Malignant glioblastomas (gliomas) account for approximately one third of all diagnosed brain tumors. Yet, a decade of research has made little progress in advancing the treatment of these tumors. In part this lack of progress is linked to the challenge of discovering how glial tumors are capable of both modulating host immune function and neutralizing immune-based therapies. Patients with gliomas exhibit a broad suppression of cell-mediated immunity. The impaired cell-mediated immunity observed in patients with gliomas appears to result from immunosuppressive factor(s) secreted by the tumor. This article reviews what has been elucidated about the immune defects of patients harboring glioma and the glioma-derived factors which mediate this immunosuppression. A model involving systemic cytokine dysregulation is presented to suggest how the immune defects arise in these individuals.

Insulin-like growth factors (IGF) enhance three-dimensional (3D) growth of human glioblastomas.

Human glioblastomas (gliomas) are characterized as rapidly growing brain tumors which are highly invasive but rarely metastatic. Human gliomas synthesize and secrete increased levels of insulin-like growth factors (IGFs) as well as expressing increased numbers of IGF receptors when compared to normal brain tissue. These observations suggest the existence of an IGF-mediated autocrine mechanism for glioma growth regulation. The purpose of this study was to examine the effect of human recombinant IGF (hrIGF) treatment on the in vitro growth of human glioma monolayer and three-dimensional (3D) multicellular spheroid cultures. The data demonstrate that hrIGF-I treatment of glioma cell lines slightly enhanced tumor monolayer proliferation as measured by [(3)H]thymidine incorporation. In contrast, treatment of glioma spheroids with hrIGF-I or hrDes(1-3)IGF-I, the truncated brain form of IGF-I, dramatically enhanced 3D tumor growth with a 1.5-2-fold reduction in spheroid doubling time (FRSDT). In addition, IGF-treated glioma spheroids were more densely packed than spheroids grown in media alone with no observed necrosis. These data suggest that IGFs will dramatically enhance glioma proliferation when 3D cell-cell contact occurs. This observed enhancement suggests that IGFs both synthesized in the brain and systemically support rapid proliferation of gliomas in vivo.

Daclizumab (Zenapax) inhibits early interleukin-2 receptor signal transduction events.

Daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor (R) alpha-chain, is a promising new immunosuppressant in transplantation. As its exact mechanism of action has remained unclear, we examined its short-term effects on primary human T lymphocytes expressing the high-affinity IL-2R. Daclizumab exposure for 20 min neither affected T cell viability nor their surface expression of the IL-2R alpha-, beta-, or gamma-chains. However, after IL-2 stimulation (200 U/ml, 20 min), immunoblots of cell lysates demonstrated attenuation of the IL-2-induced tyrosine phosphorylation of 65-75 kDa proteins by Daclizumab, but not by isotype controls. Since this is the molecular weight of the IL-2R beta- and gamma-chains, which are both tyrosine-phosphorylated by IL-2, we next examined the effect of Daclizumab on their IL-2-induced tyrosine phosphorylation. In immunoblots of IL-2R beta- and gamma-chain-immunoprecipitates the tyrosine phosphorylation of both chains by IL-2, but not by IL-15, was attenuated in the presence of Daclizumab. Furthermore, co-immunoprecipitation experiments showed that Daclizumab inhibited the IL-2-induced association of these chains, a prerequisite for their mutual tyrosine phosphorylation. Lastly, we demonstrated that Daclizumab inhibits the receptor-downstream induction of the IL-2-activated DNA-binding protein STAT5 in gel shift assays. We conclude that Daclizumab directly and specifically interferes with IL-2 signaling at the receptor level by inhibiting the association and subsequent phosphorylation of the IL-2R beta- and gamma-chains induced by ligand binding. Under our experimental conditions, Daclizumab had no effects on cell viability, and it did not modulate the surface expression of the IL-2R alpha-, beta-, or gamma-chains.

Hypothalamic-immune interactions. I. The acute effect of anterior hypothalamic lesions on the immune response.

Rats with electrolytic anterior hypothalamic lesions show changes in lymphoid tissue cellularity and a decrease in the response to concanavalin A (Con A). This effect manifests itself maximally 4 days after lesioning, with a return to normal by day 14. The changes are not mediated through the release of corticosteroids. These data indicate the presence of a neuroendocrine pathway that is capable of modulating immune function.

Hypothalamic-immune interactions. Effect of hypophysectomy on neuroimmunomodulation.

Electrolyte destruction of certain nuclei of the brain cause specific structural and functional changes in the immune system. Lesions in the preoptic-anterior hypothalamic area result in thymic involution and a decrease in the number and blastogenic reactivity of splenocytes. In contrast, lesions in the hippocampus increase thymic and splenic mitogenic responsiveness and cellularity. Hypophysectomy abrogates all changes in splenocyte number and function induced by hypothalamic and limbic lesions. The effects of ablating the hippocampus and amygdaloid complex on thymocyte number and function also are abolished. Hypothalamic lesions in hypophysectomized animals result in an increase in the number of thymocytes but suppressed mitogenic activity. These data indicated that neuroimmunomodulation is mediated predominantly but not exclusively by the pituitary gland.

Inhibition of lymphocyte responsiveness by a glial tumor cell-derived suppressive factor.

The results of this study demonstrate the presence of suppressive factor(s) in the tissue culture supernatants of cloned and freshly explanted malignant glioma cells. Culture supernatants obtained from these glial cell lines were demonstrated to have potent suppressive activity as evidenced by their ability to inhibit the proliferative response of normal human peripheral blood lymphocytes induced by phytohemagglutinin and anti-OKT3 monoclonal antibodies. The results further demonstrate the existence of a dose-response relationship between these supernatants and inhibition of mitogen-induced lymphocyte activation. Maximum production of suppressive activity by glial tumor cells was dependent on: 1) the number of tumor cells seeded in culture, 2) whether fetal calf serum was present, and 3) the duration of culture. The production of the suppressive factor(s) was not inhibited by the addition of inhibitors of prostaglandin E synthesis. Experiments designed to determine at what time during lymphocyte activation the suppressive factor was most effective demonstrated that the culture supernatants must be added during the first 24 hours of culture to exhibit inhibitory properties. Finally, proliferation of both the T-helper and T-suppressor/cytotoxic subsets was equally well inhibited by the glial tumor cell culture supernatants.

Activation of immunoregulatory lymphocytes obtained from patients with malignant gliomas.

The responsiveness of T cells and their subsets (T-helper cells and T-suppressor cells) obtained from patients with malignant gliomas was evaluated in an effort to further define the mechanism of their impaired host immunocompetence. This study demonstrates that peripheral blood lymphocytes obtained from these patients have impaired responsiveness to a variety of mitogens including phytohemagglutinin, concanavalin A, pokeweed mitogen, and anti-T3 monoclonal antibody. The impaired lymphocyte responsiveness does not result from the inability of these cells to express receptors for a specific mitogen or antibody. The mitogenic responsiveness of purified T cells is markedly reduced when compared to values obtained from control subjects. Therefore, the decreased T cell reactivity of patients with malignant gliomas does not result simply from a diminution in the absolute number of potentially responding lymphocytes. The mitogen reactivity of the T cell subsets, CD4+ helper cells, and CD8+ cytotoxic/suppressor cells was also investigated. These results demonstrate that the responsiveness of the CD4+ T-helper cell subset obtained from these patients is consistently diminished as compared to control values. In contrast, the reactivity of the CD8+ T cell subset was not nearly as dramatically impaired. Thus, these results indicate that the proliferative defect observed in T cells obtained from patients is located predominantly in the T-helper cell subset. Functional deficiencies in this important subpopulation of T lymphocytes may explain, in part, the presence of depressed immune responsiveness in patients with malignant glial tumors.

Immunobiology of primary intracranial tumors. Part 1: studies of the cellular and humoral general immune competence of brain-tumor patients.

Cellular and humoral immune parameters were evaluated in a series of brain-tumor patients, 42 with glioblastoma multiforme, 17 with other anaplastic gliomas, and 17 with meningiomas. A degree of anergy was found, which seems in the group as a whole to be proportional to the degree of anaplasia. In addition, serial bimonthyl testing in individual cases revealed further reduction in certain immune responses coincident with clinical decline.