Hypophysectomy and neurointermediate pituitary lobectomy decrease humoral immune responses to T-independent and T-dependent antigens.

In rats, hypophysectomy (HYPOX) or neurointermediate pituitary lobectomy (NIL) reduce humoral and cell-mediated immune responses. However, to our knowledge, the differences in the effects of anterior versus posterior pituitary hormones on the immune responses have not been studied to date. We compared in rats, the effects of sham surgery (SHAM), HYPOX, and NIL on humoral immune responses to T cell-independent (TI) type 1 antigen DNP-LPS and to TI type 2 antigen DNP-FICOLL, as well as to T cell-dependent (TD) antigens ovalbumin (OVA) and bovine serum albumin (BSA). The results showed that: (1) both HYPOX and NIL induced a similar and significant decrease in IgM responses towards TI-1 antigens, (2) NIL but not HYPOX induced a decreased IgM response to TI-2 antigens, and (3) both HYPOX and NIL induced similar and significant decrease in IgG responses to TI-2 antigens. Compared with the SHAM group, IgM responses to both TD antigens did not change in HYPOX and NIL animals, whereas the IgG responses to OVA and BSA significantly decreased in HYPOX and NIL animals. These results indicate that hormones of the anterior and posterior pituitary play their own role in the regulation of humoral immune responses.

Antiestrogens affect both pathways of killer cell-mediated oncolysis.

BACKGROUND: Our previous studies indicate that antiestrogenic drugs tamoxifen (TX) and toremifene (TO) augment immune oncolysis induced by various killer cells. The underlying mechanism(s), however, have not been fully elucidated. MATERIALS AND METHODS: Ovarian carcinoma cells freshly isolated from cancer patients and the human erythroleukemia cell line, K562 were used as targets for killer cells and/or the anti-Fas monoclonal antibody, CH-11 in 51Cr release assays. In a number of experiments, extracellular Ca++ was chelated by EGTA/MgCl2 to distinguish Ca(++)-dependent perforin/granzyme pathway from Fas/FasL pathway. Fas expression was studied by flow cytometry. RESULTS: Ovarian carcinoma cells were sensitized by antiestrogens towards enhanced cytolysis mediated by autologous cytotoxic lymphocytes. Antiestrogens also significantly augmented the killing of ovarian carcinoma cells triggered by anti-Fas monoclonal antibody. Flow cytometry analyses showed an upregulation of Fas (CD 95/Apo-1) upon TX or TO treatment in a number of cases. By contrast, antiestrogen treatment did not induce Fas expression in the Fas-negative K562 cells; yet, natural killer cell-mediated cytotoxicity against K562 was augmented by antiestrogens and maximal lysis was achieved when both target and effector cells were treated. The presence of Ca++ chelator (EGTA/MgCl2) in the assay abrogated killing of K562 and its antiestrogen--mediated augmentation. This indicates the involvement of the perforin/granzyme pathway. CONCLUSION: Antiestrogens can influence both Fas/FasL and perforin/granzyme pathways of killer cell--mediated oncolysis.

Antiestrogens sensitize human ovarian and lung carcinomas for lysis by autologous killer cells.

BACKGROUND: The antiestrogens tamoxifen (TX) and toremifene (TO) were shown previously to enhance the lysis of target cells by natural killer cells (NK), lymphokine activated killer (LAK) cells, and by cytotoxic T lymphocytes (CTL). MATERIALS AND METHODS: CTL were cultured from lung cancer tissue and from ascites fluid of ovarian carcinoma patients with the aid of human recombinant interleukin-2 (hrIL-2). The target, effector or both cell populations were pretreated by TX, TO and/or with human recombinant interferon-alpha (IFN-alpha). RESULTS: Significant enhancement of cytotoxicity occurred when the tumor targets or both the target and effector cells were treated with TX, TO or when these drugs were used in combination with IFN-alpha. The lytic activity of CTL cultured from draining lymph nodes of lung cancer patients, was also observed after similar treatment. The lytic effect of autologous LAK cells derived from peripheral blood was increased to a lesser extent, which could be amplified by additional treatment with IFN-alpha. CONCLUSIONS: The antiestrogens TX and TO and IFN-alpha enhance the lysis of autologous tumor cells by CTL and LAK effectors.

Suppression of lymphocyte mitogenesis by tamoxifen: studies on protein kinase C, calmodulin and calcium.

The effect of tamoxifen (TX; 1.0 microM) on the mitogenic response of rat lymphocytes was compared with the effect of drugs that are known to act on protein kinase C (PKC), calmodulin (CM), and calcium (Ca(2+)). The calcium ionophore A23187 (0.2 microM) was mitogenic on its own which was not influenced by TX. The agents modulating PKC or CM (phorbol-myristate-13-acetate; R24571, chlorpromazine) influenced mitogenesis differently than did TX. General inhibition of lymphocyte proliferation was seen with the Ca(2+) antagonist agents (EGTA, TMB-8) as with TX. The antiproliferative effect of TX was partially reversed by the increase of Ca(2+) in the culture medium when T cell mitogens were used, but not in the case of lipid A, a B lymphocyte mitogen. However, the concanavalin A-induced Ca(2+) influx was further elevated by TX which differed from the effect of the Ca(2+) channel-blocking agent verapamil. The results suggest that TX resets the threshold stimulus necessary for mitogenesis and is completely reversible.

Natural immunity and neuroimmune host defense.

Innate resistance is mediated by non-immune defense and by natural immunity. Non-immune defense includes diverse mechanisms (e.g., physico-chemical defense by bile acids). Natural killer (NK) cells, gamma delta T lymphocytes and CD5+ B lymphocytes are key mediators of natural immunity. These cells utilize germ-line coded receptors that recognize highly conserved, homologous epitopes (homotopes). Typically, it is not the antigen, but cytokines and hormones that regulate the level of NK-mediated cytotoxicity. These include interleukin-2, interferons, prolactin and growth hormone. Less is known about gamma delta T lymphocytes. CD5+ B lymphocytes produce germ-line coded antibodies (predominantly IgM) that are polyspecific, and able to recognize a great variety of microorganisms, cancer-cells and self-components. Antigen is not an effective stimulus for natural antibody (NAb), but bacterial lipopolysaccharide (LPS) is. During the acute phase response (febrile illness) the T-cell-regulated adaptive immune response is switched off and natural immune mechanisms are amplified several hundred to a thousand times within 24-48 hours (immunoconversion). This immunoconversion is initiated by immune-derived cytokines, and involves profound neuroendocrine and metabolic changes, all in the interest of host defense. Immune recognition is assured by natural antibodies and by some liver-derived acute phase proteins, such as C-reactive protein or endotoxin-binding protein, the level of which is elevated in the serum. Thus, natural immunity is essential for a first and last line of defense and the neuroendocrine system is an important promoter of this activity.

Neuroimmunoregulation and cancer (review).

It is certain that neuroimmune mechanisms play a role in host defence against cancer. However, this interaction is highly complex and many variations are possible according to the nature of the neoplasms involved. There are indications that adaptive immunity is present in a significant proportion of tumor bearing hosts, and this defence may be boosted by specially designed vaccines and cytokines. Natural immune mediators are also implicated in resistance against tumor development. Here we review the evidence suggesting that hormonal manipulation of the host can result in the elevation of immune defences against cancer. Such manipulation strengthens both the adaptive and natural immune defences of the host, both of which play significant roles. Natural defence mechanisms are boosted by cytokines and hormones during febrile reactions which are now known as the acute phase response. It is suggested that hormonal stimulation of immune mechanisms coupled with the usual immunostimulants already in use may be employed to good advantage for the combination immunotherapy of cancer. Modern molecular biology approaches permit the development of laboratory monitoring procedures which may be used for the prediction and follow-up of therapeutic success.

Combination therapy of the H2712 murine mammary carcinoma with cytotoxic T lymphocytes and anti-estrogens.

BACKGROUND: The triphenylethylene antiestrogenic agents, tamoxifen (TX) and toremifene (TO), are currently being used for the therapy of estrogen dependent breast carcinomas and for some other estrogen receptor positive tumors. Some observations indicate that these drugs may have a beneficial effect on estrogen receptor negative tumors as well. MATERIALS AND METHODS: The H2712 mammary carcinoma of C3H/HeJ mice was studied in combination immunotherapy experiments using cytotoxic T lymphocytes (CTL) and TX or TO. The effect of TX and TO on the in vitro lysis of H2712 cells by CTL was also investigated. Finally, the H2712 cells were examined for the presence of estrogen receptors. RESULTS: Both TX and TO potentiated the lysis of H2712 cells by CTL in vitro, and exerted a growth inhibitory and therapeutic effect on H2712 mammary carcinomas in vivo. The therapeutic effect of CTL isolated from tumor bearing mice was improved on H2712 carcinomas when the animals were also treated orally by TX or TO. Using the dextran-coated charcoal assay, H2712 cells were found to be negative for classical estrogen receptors. CONCLUSIONS: These results indicate that the anti-estrogens, TX and TO, have the ability to suppress the growth of the estrogen receptor negative mammary carcinoma and to amplify target cell lysis by tumor-reactive CTL. By this mechanism these drugs enhance host immunity to an estrogen receptor negative tumor.

Combination immunotherapy of the P815 murine mastocytoma with killer cells, IL-2 and anti-estrogens.

BACKGROUND: Lymphokine activated killer (LAK) cells, cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2) all are being tested in cancer immunotherapy with modest success. The anti-estrogenic drug, toremifene (T0), was found earlier to amplify cancer immunotherapy with killer cells in mice. Here T0 is examined in combination with CTL and IL-2 in immunotherapy experiments. MATERIALS AND METHODS: The P815 mastocytoma of DBA/2 mice was treated with combinations of CTL, LAK cells and T0 and combinations of CTL, IL-2 and T0 along with control groups. Tumor growth rate and mortality has been recorded. RESULTS: Combined treatment with CTL and LAK cells cured 25% of tumor bearing animals, as did treatment with tamoxifen (TX) or T0 alone. When killer cells were given in conjunction with anti-estrogens the cure rate was 75% with both TX and T0. Human recombinant IL-2 significantly inhibited tumor growth but no cures occurred. CTL alone cured 25% of the animals. When CTL and IL-2 treatment was given jointly with T0 75% of tumor bearing animals were cured. CONCLUSIONS: The results indicate that treatment with anti-estrogens increased the efficiency of immunotherapy by killer cells. This was true also when CTL therapy was supplemented with IL-2 treatment.

Anti-estrogens potentiate the immunotherapy of the P815 murine mastocytoma by cytotoxic T lymphocytes.

Many animal and human tumors are infiltrated with killer cells. Recent studies have shown that the stimulation of such killer cells with interleukin-2 improves their tumor rejecting capacity. In this paper we demonstrate that the anti-estrogens, tamoxifen (TX) and toremifene (TO), enhance host resistance by sensitizing the tumor target to killer cell mediated lysis. The P815 mastocytoma syngeneic to DBA/21 mice was used. Cytotoxic T lymphocytes (CTL) were detected in the spleens of mice 12-14 days after tumor inoculation. In vitro target, effector, or both cell types were treated with either TX (1 microM) or TO (5 microM) for 4 hours prior to cytotoxicity testing by 51Cr release. Mice bearing P815 mastocytomas, 5 mm in diameter, were treated orally with TX or TO and were given CTL isolated from the spleens of tumor bearing donors i.p. Tumor growth, mortality, and immunological memory in cured animals were monitored. Both TX and TO treatment sensitized P815 target cells to lysis by CTL isolated from tumor bearing animals. The transfer of killer cells from the spleens of tumor bearing mice produced tumor suppression in the recipients, which could be enhanced by additional oral treatment by TX or TO. Complete cure was achieved in a significant number of animals, showing partial or complete resistance to a subsequent lethal dose of P815 cells. These experiments indicate that killer cells isolated from mice bearing progressive tumors can have an immunotherapeutic effect in syngeneic tumor bearing recipients and that the antiestrogens, TX and TO, may be used to potentiate the immunotherapy of this tumor.

Immunotherapy of the SL2-5 murine lymphoma with natural killer cells and tamoxifen or toremifene.

It is well established that tamoxifen (TX) has a therapeutic effect on estrogen receptor positive tumors by inhibiting the binding of estradiol to its receptor. However, repeated clinical observations indicate that tamoxifen may also have beneficial effects on estrogen receptor negative tumors. In vitro cytotoxicity experiments were performed with the SL2-5 murine lymphoma using interleukin (IL)-2 activated syngeneic NK cells as effectors with or without TX or TO treatment. The effect of TX or TO (10 mg/kg/day/animal in feed) on the immunotherapy of SL2-5 lymphoma with syngeneic IL-2 activated NK cells was also investigated in syngeneic DBA/2 mice. Assays of SL2-5 cells for estrogen and progesterone receptors were also performed. Both TX and TO enhanced significantly the susceptibility of the SL2-5 lymphoma to lysis by IL-2 activated NK cells in vitro. When TX or TO treatment was combined with NK cell immunotherapy of this tumor, both drugs potentiated significantly tumor regression and cure rate when compared to groups receiving NK therapy alone. This tumor does not express classical receptors for estrogens or progesterone. These results indicate that combination treatment with the antiestrogens, TX and TO, acts synergistically with the immunotherapeutic effect of IL-2 activated NK cells in a syngeneic tumor host system.

Neuroimmune mechanisms in health and disease: 2. Disease.

In the second part of their article on the emerging field of neuroimmunology, the authors present an overview of the role of neuroimmune mechanisms in defence against infectious diseases and in immune disorders. During acute febrile illness, immune-derived cytokines initiate an acute phase response, which is characterized by fever, inactivity, fatigue, anorexia and catabolism. Profound neuroendocrine and metabolic changes take place: acute phase proteins are produced in the liver, bone marrow function and the metabolic activity of leukocytes are greatly increased, and specific immune reactivity is suppressed. Defects in regulatory processes, which are fundamental to immune disorders and inflammatory diseases, may lie in the immune system, the neuro endocrine system or both. Defects in the hypothalamus-pituitary-adrenal axis have been observed in autoimmune and rheumatic diseases, chronic inflammatory disease, chronic fatigue syndrome and fibromyalgia. Prolactin levels are often elevated in patients with systemic lupus erythematosus and other autoimmune diseases, whereas the bioactivity of prolactin is decreased in patients with rheumatoid arthritis. Levels of sex hormones and thyroid hormone are decreased during severe inflammatory disease. Defective neural regulation of inflammation likely plays a pathogenic role in allergy and asthma, in the symmetrical form of rheumatoid arthritis and in gastrointestinal inflammatory disease. A better understanding of neuroimmunoregulation holds the promise of new approaches to the treatment of immune and inflammatory diseases with the use of hormones, neurotransmitters, neuropeptides and drugs that modulate these newly recognized immune regulators.

Anti-estrogens enhance the therapeutic effect of lymphokine-activated killer cells on the P815 murine mastocytoma.

Tamoxifen (TX) and toremifene (TO) enhanced the lysis of P815 mastocytoma cells in vitro by syngeneic DBA2 spleen cells that have been activated by human recombinant interleukin-2 (IL-2) for 6 days (lymphokine-activated killer [LAK] cells). Similarly, enhanced tumor suppression occurred when TX- or TO-treated P815 cells were mixed with LAK cells and injected s.c. into normal DBA2 recipients. Tumor suppression could be increased further by treating such recipients orally with TX or TO and by the repeated injections of LAK cells into the tumor site. The treatment of animals bearing tumors (5 mm in diameter) orally with TX or TO or with LAK cells i.p. resulted in tumor suppression. When the drug treatment was combined with LAK cells, tumor suppression was more pronounced, and complete tumor regression was induced in a significant number of the animals so treated. Our results indicate that the immunotherapeutic effect of LAK cells can be significantly amplified by combined treatment with the anti-estrogens TX or TO.

Modulation of lymphokine-activated killer cell-mediated cytotoxicity by estradiol and tamoxifen.

The effect of tamoxifen (TX) and estradiol (E2) on interleukin-2 (IL-2 )-activated killer (LAK) cell-mediated cytotoxicity was examined using spleen cells of Fischer 344 rats as the source of effectors and P815 murine mastocytoma cells as targets. Treatment of target cells with either TX or E2 for 4 or 18 hr rendered them highly sensitive to LAK cell-mediated lysis. When TX and E2 were applied jointly, cytotoxicity remained at the level of TX alone. The cytotoxic potential of IL-2-primed LAK cells was not modified consistently by TX and E2. When TX-treated target and effector cells were combined, high cytotoxicity characteristic of sensitized target cells was observed. In similar experiments with E2-treated cells, both enhancement and inhibition of cytotoxicity by treated effector cells was seen in some designs. Target cells could be sensitized for LAK cell-mediated destruction by physiological concentrations (1 nM) of E2 and equimolar concentration of TX. Sensitization led to the accelerated release of the nuclear label 3H-thymidine from target cells after cytotoxic insult and could be prevented by treatment with the metabolic inhibitors cycloheximide and actinomycin D. Enhanced 3H-thymidine release from TX-treated targets was also demonstrated after induction of Ca2+ influx by exposure to the ionophore A23187. Neither E2 nor TX exerted a direct cytotoxic effect on P815 cells. P815 cells had no classical receptors for E2 or progesterone.

The submandibular gland: a key organ in the neuro-immuno-regulatory network?

The evidence for the integration of the submandibular gland (SMG) into the neuroimmunoregulatory network has been reviewed. In laboratory rodents, factors extracted from the SMG were shown to stimulate lymphocyte proliferation, to affect the weight of the thymus, spleen and lymph nodes and to induce immunosuppression in several in vivo animal models. The SMG produces significant quantities of nerve growth factor (NGF), epidermal growth factor (EGF), transforming growth factor-beta and kallikreins, which are secreted into the saliva and affect immune and mucosal tissues and nerve endings in the gastrointestinal tract. These factors play a role in regulating mucosal immuno/inflammatory response and in regeneration and healing. The major salivary glands also produce antimicrobial proteins and secretory IgA antibodies which are essential factors in mucosal host defense. SMG-derived NGF, EGF and glandular kallikrein are delivered into the bloodstream where they may act as important systemic immunoregulators and also have major regulatory influences on the central neuroendocrine system. There is evidence to indicate that EGF is involved in the regulation of gonadal function. Growth hormone, prolactin, androgens, thyroid hormone and corticosteroids regulate protein synthesis in the SMG, whereas secretory activity is regulated by sympathetic (alpha- and beta-adrenergic) parasympathetic (muscarinic) and peptidergic (substance P and vasoactive intestinal peptide) nerve fibers. Fluid and electrolyte secretion is promoted by parasympathetic, whereas protein secretion is stimulated by sympathetic nerve impulses. Steroid hormones and cytokines (interleukin-1 alpha, -beta, tumor necrosis factor, interferon-gamma) have a major regulatory influence on protein secretion, including the secretion of immunoglobulin into the saliva. The SMG interacts with the mucosal and systemic compartments of the immune system, with the central and peripheral nervous systems, with the pituitary gland, and with peripheral endocrine organs. These interactions enable the SMG to exert regulatory influences on immune/inflammatory reactions in the gastrointestinal tract, in the lungs, and possibly elsewhere. It is suggested that these functions make this gland a key regulatory organ in the neuroimmunoregulatory network. Evidence is increasing that the major salivary glands fulfill similar functions in other species, including humans.

Modulation of natural killer cell-mediated cytotoxicity by tamoxifen and estradiol.

BACKGROUND: The nonsteroidal antiestrogenic drug, tamoxifen, inhibits the growth of estrogen receptor-positive tumors by interfering with the growth-stimulatory effect of estradiol. However, there is compelling evidence that tamoxifen treatment also is beneficial for patients with estrogen receptor-negative tumors. The hypothesis that tamoxifen is capable of enhancing the immunologic defense of tumor-bearing hosts was been investigated as a possible method for targeting receptor-negative neoplasms. METHODS: Natural killer (NK) cells in the spleen of female Fisher and Wistar-Furth rats were used against the YAC-1 murine lymphoma target in 51Cr-release assays. The effect of various concentrations of estradiol and tamoxifen (1 nM-1 microM) and of the metabolic inhibitors actinomycin D and cyclohexamide on target-cell killing was investigated. RESULTS: Tamoxifen enhanced and estradiol inhibited killing if applied for the entire 5 hours of the cytotoxic reaction. When applied jointly in this experimental setup, estradiol interfered with the enhancing effect of tamoxifen. Pretreatment of target cells with tamoxifen led to highly significant enhancement of cytotoxicity; estradiol also enhanced target cell killing, but to a lesser extent. After joint treatment, the level of cytotoxicity was comparable with that obtained with tamoxifen alone. Both pharmacologic (100 nM and 1 microM) and physiologic (1 or 10 nM) concentrations of estradiol and equimolar tamoxifen enhanced target cell lysis. However, pharmacological levels of estradiol inhibited effector cells when applied alone or in combination with tamoxifen. Highly significant enhancement of target-cell destruction occurred if both target and effector cells were pretreated with tamoxifen, whereas estradiol treatment of both cell types led to slight enhancement or no effect on cytotoxicity. Treatment of the target cells with actinomycin D or cycloheximide inhibited the lysis of untreated and tamoxifen- or estradiol-exposed cells. Treatment of YAC-1 target cells with tamoxifen or estradiol also enhanced the NK cell-mediated release of the nuclear label, 3H-thymidine, indicating DNA degradation. Similarly treated P815 cells resisted lysis by NK cells, but showed sensitization when the NK cells were stimulated by interleukin-2 for 48 hours before the lytic reaction. Estradiol and tamoxifen changed the kinetics of 3H-thymidine incorporation by YAC-1 cells, but the cells were capable of growing with the highest drug concentrations (1 microM) used in the cytotoxicity experiments. YAC-1 cells have no cytosolic estradiol receptors and are weakly positive for cytosolic progesterone receptors. CONCLUSIONS: These experiments indicate that NK cell-mediated target-cell destruction can be enhanced by tamoxifen primarily through sensitizing the target for lysis. Estradiol also sensitizes the target but inhibits the effector cells simultaneously so that little or no change results in cytolysis. Target-cell sensitization is not mediated by classical estrogen receptors and requires the active metabolic participation of the cells treated. A likely mechanism of this phenomenon is priming the target cell for apoptosis.

Target cells are sensitized for cytotoxic T-lymphocyte-mediated destruction by estradiol and tamoxifen.

The effect of estradiol (E2) and tamoxifen (TX) on cytotoxic T-lymphocyte (CTL)-mediated target-cell lysis was studied. CTL was generated in mixed cultures of rat spleen cells, using female Fischer 344 rat cells as responders and female Wistar rat cells or Nb2 rat lymphoma cells as stimulators. Concanavalin-A-stimulated Wistar lymphoblasts or Nb2 cells were used as targets. CTL harvested on day 5 exerted 16-25% cytotoxicity when used at 1:12-1:50 target:effector cell ratios. Day-6 CTL had no cytotoxic activity. Treatment of target cells with either TX or E2, or both, at 1-microM concentrations for 4 hr prior to cytotoxicity testing raised the target-cell killing to 100%. Highly significant enhancement of cytotoxicity was also observed when the drugs were used at 100-, 10-, or 1-nM concentrations. Treatment of effector cells under similar conditions led to inhibition of cytotoxicity at 1-microM concentration, some enhancement at 100 nM and no effect at 10 and 1 nM. When treated target and effector cells were combined, the amplification of target-cell lysis was similar in magnitude to that seen in tests with treated targets only. Drug treatment of target cells had no influence on their resistance to lysis in hypotonic solutions or on total and spontaneous 51Cr release. The inhibition of DNA and protein synthesis in target cells interfered with both basal cytolysis and drug-induced enhancement. The release of the nuclear label, [3H]-thymidine, from Nb2 targets by CTL, was also enhanced by target treatment with TX. These results illustrate that CTL-mediated cytotoxicity is amplified by physiological concentrations (1 and 10 nM) of E2 and equimolar concentrations of TX.

The role of the growth and lactogenic hormone family in immune function.

The influence of various members of the growth and lactogenic hormone family on the immune system is reviewed. A general hypothesis is proposed for growth control in higher animals. It is suggested that immune reactions, which are based on lymphocyte proliferation, obey the general rules of growth control in vertebrate animals. Growth and lactogenic hormones (GLH) are required for the development and function of the immune system and are suggested to deliver the first signal that prepares the cell for proliferation, differentiation and function. This signal has already been designated by other as the competence signal which initiates the cell cycle. Second signals are delivered through the antigen receptor, and/or by some other cell surface receptors (adhesion molecules) and always involve cell-to-cell ('bridging') and/or cell-to-matrix interaction. This category of signals is designated as stromal or adherence signals. The lymphocyte adhesion molecules that mediate second signals have evolved form organ- and tissue-specific recognition/regulatory molecules. The antigen receptors have been perfected during evolution from self recognition to specific-antigen recognition. Apart from this exquisitely specific mechanism of immune recognition, there is evidence for other less specific means of recognition by adherence molecules that mediate the activation of the immune system during nonspecific injury and also play a role in the elimination of degenerated and neoplastic cells. Signals delivered through adhesion molecules have the power to commit the cell to a given activity which is executed by the delivery of third signals in the form of soluble cytokines, usually, but not always, by the same cell delivering the second signal(s). The combination of these three groups of signals will ultimately determine whether or not the cell will proliferate, differentiate, maintain function or, perhaps, be committed to apoptosis. Therefore, GLH maintain immunocompetence which enables the immune system to respond to specific antigenic and tissue-derived stimuli in a self-regulated fashion. The adrenocorticotropic hormone-adrenal axis antagonizes the immunostimulatory effect of GLH. This basic pattern of lymphocyte regulation is influenced further by additional hormones, neurotransmitters and neuropeptides, mostly by the modulation of signal delivery. The constant interaction of neuroendocrine and internal immunoregulatory mechanisms assures the fine tuning of the immune system, so that it is able to function in homeostasis and harmony with the organism.

Production and characterization of monoclonal antibodies to embryo-associated immunosuppressor factor (EASF) produced by human pre-implantation embryo.

Balb/c mice were immunized with pre-implantation embryo-associated immunosuppressor factor (EASF) (purified from embryo growth media of in vitro fertilized human ova). Hybridoma clones were produced by fusing their spleen cells with NS1 and P3X653 myeloma cell lines. The presence of specific anti-EASF monoclonal antibodies (mAb) in the hybridoma culture supernatants were tested by enzyme-linked immunosorbent assay. A total of 15 hybridoma clones were selected, and their products were purified and characterized. Each mAb bound specifically to its antigen in a dose-dependent manner. The affinity-purified EASF from embryo growth media demonstrated immunosuppressive activity on Concanavalin A-induced lymphocytes and the presence of 14 kDa, 24 kDa and 37 kDa factors. No such activity or similar molecules were identified when control growth media were analyzed. This clearly demonstrates that these mAb are indeed EASF-specific and are able to recognize biologically active immunosuppressive components in embryo growth media. These mAbs are presently being tested for the development of EASF-specific assay system.

Inhibition of lipid A- and lipopolysaccharide-induced cytokine secretion, B cell mitogenesis, and lethal shock by lipid A-specific murine monoclonal antibodies.

Three murine hybridomas secreting IgM monoclonal antibodies (MAbs) to lipid A (LA) of Salmonella minnesota R595 were generated. These MAbs serologically cross-reacted with LA and lipopolysaccharide (LPS) of unrelated gram-negative bacterial species. All three MAbs significantly suppressed the ability of LA and LPS from various gram-negative bacteria to induce tumor necrosis factor (TNF)-alpha (36%-67%) and interleukin-1 (30%-98%) in murine peritoneal macrophages and to stimulate B lymphocytes (37%-78%). Lipid A-induced TNF alpha production was also suppressed in mice (86%-88%). All three antibodies protected adrenalectomized mice against lethal shock induced by LA of S. minnesota R595. Optimal protection was achieved with one of the antibodies (MLA-1), if it was administered 2 h before injection of lipid A, and full protection persisted < or = 24 h. Moreover, MLA-1 was able to protect adrenalized or D(+)-galactosamine-sensitized mice against lethal shock induced by LPS derived from various gram-negative bacteria. This cross-protection could be predicted on the basis of serologic cross-reactivity and cross-neutralization by MLA-1 of the bioactivity of the heterologous LA or LPS in vitro.

Endocrine control of the immunosuppressive activity of the submandibular gland.

Extracts of the submandibular gland (SMG) of rats contain fractions that stimulate the in vitro proliferation of Con A-treated lymphocytes. One of the stimulatory fractions was also shown to induce in vivo immunosuppression in rats and mice in several experimental models. Since many other biologically active factors of the SMG had been found to be hormone dependent, we investigated the effects on the immunosuppressive factor of hypophysectomy (Hx) and of hormonal reconstitution in male Fischer rats. Hx induced a marked atrophy of the SMG together with an almost complete disappearance of both the in vitro lymphocyte-stimulating activity and the in vivo immunosuppressive activity, the latter assayed with the contact sensitivity reaction in mice. The treatment of the Hx rats with pituitary hormones demonstrated that prolactin (PRL), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) induced a significant reconstitution of these biological activities, growth hormone led to the recovery of the lymphocyte-stimulating activity but not of the immunosuppressive activity, while follicle-stimulating hormone, and adrenocorticotropic hormone did not induce any recovery of these biological activities. In view of the positive results obtained with TSH and LH further experiments were done to compare the effects of thyroid and sex hormones with those of PRL. The results demonstrated that testosterone and thyroid hormones induced significant recovery of the lymphocyte-stimulating and the immunosuppressive activity. The combination of these two hormones with PRL produced the most effective results. On the other hand, estrogens and progesterone had no significant effects. These results confirm the effectiveness of androgens and thyroid hormones in stimulating the production of biologically active factors by the SMG. Moreover, they demonstrate that PRL, a hormone not previously considered to increase the activity of the SMG, stimulates the production of immunoregulatory factors in Hx animals.