Elba M. Hadden

Zinc induces thymulin secretion from human thymic epithelial cells in vitro and augments splenocyte and thymocyte responses in vivo.

Zinc is incorporated into zinc-thymulin by the thymus and in this form is a critical hormonal regulator of cellular immunity. In the absence of serum, zinc induces human thymic epithelial cells (TEC) to secrete a factor which promotes the expansion of interleukin-2 (IL-2) receptor positive human peripheral blood lymphocytes in response to a low dose of phytohemagglutinin (PHA). This factor is removed by antithymulin antisera plus filtration and is thus presumed to be zinc-thymulin. Intraperitoneal treatment of hydrocortisone treated aged mice with zinc-thymulin (100 ng/day x 5) resulted in mild augmentation of splenocyte but not thymocyte responses in vitro to IL-1, IL-2, and natural cytokine mixture (NCM) and to PHA and concanavalin A (Con A) (average increase 40%). Like zinc-thymulin treatment, oral ingestion of zinc (72 micrograms/day x 5) resulted in augmentation of splenocyte IL responses; in contrast, it augmented thymocyte responses to all stimuli (average increase 100%). These preliminary experiments indicate that treatment with zinc may have immunotherapeutic relevance, particularly in the aged and stressed organism.

Immunotherapy with natural interleukins and/or thymosin α1 potently augments T-lymphocyte responses of hydrocortisone-treated aged mice

Cytokines and thymic hormones are thought to play critical roles in the regulation of T-lymphocyte development and function. In an effort to determine the effectiveness of such agents in an immunotherapeutic strategy, we employed aged mice in a hydrocortisone treatment model to generate an immunodeficient state and to study its reconstitution. Mice were given five daily injections of a natural cytokine mixture (NCM), recombinant interleukins (rIL-1, rIL-2) or their combination, thymosin alpha 1 or fraction 5 (T alpha 1, TF5), or the combinations of NCM plus T alpha 1 and of NCM plus TF5. Spleen and thymus weights were obtained and the cellular responses to stimulation in vitro with NCM, IL-1, IL-2 and mitogens (PHA and Con A) were assayed. Both NCM and T alpha 1 in vivo treatment augmented thymocyte and splenocyte in vitro responses to both interleukins and mitogens. Neither treatments with equivalent doses of rIL-1, rIL-2 nor their combination, nor TF5 achieved similar results. Of all the treatments, only NCM plus T alpha 1 augmented spleen weight; none augmented thymus weight. Surface marker analyses of T-lymphocytes and subsets indicate that treatment of mice with NCM plus T alpha 1 increased spleen T-cell numbers of both CD4 and CD8 positive cells significantly. These data indicate that NCM and T alpha 1 alone and in combination may be therapeutically useful to restore T-lymphocyte number or function in secondary immunodeficiency.

Mixed interleukins and thymosin fraction V synergistically induce T lymphocyte development in hydrocortisone-treated aged mice

Analysis of the role of interleukins in T cell ontogeny in vitro indicates that the regulation of T cell development involves interleukins (ILs) as well as thymic hormones (THs). In order to assess their respective roles in T lymphocyte development in vivo, chemically thymectomized mice were treated with ILs and THs. After 2 days of hydrocortisone treatment, aged mice showed acute thymic involution (weight was less than 30% of control) and reduced spleen size (less than 80% of control) with progressive recovery to 8 days. After 2 days of hydrocortisone treatment, adult mice were injected for 5 days with mixed buffy coat interleukins (BC-IL; 50 units IL2 equivalence), purified IL2 (50 units), rIL1 beta (4 ng), and thymosin fraction V (TF5; 100 micrograms). The animals were sacrificed and spleens and thymuses were analyzed for weight, cellularity, T cell number, subsets, and function as determined by proliferative responses to concanavalin A and ILs. BC-IL treatment increased the recovery of spleen and thymus weights and cellularity with corresponding augmentation of number and function of T lymphocytes; neither IL1 or IL2 or their combination had this effect. TF5 had no effect alone but strongly potentiated the effect of BC-IL on T lymphocyte function. These data indicate that BC-IL in combination with thymic peptides potently promotes T lymphocyte development. The combination may be therapeutically relevant for immunorestoration.

Prolactin and known modulators of rat splenocytes activate nuclear protein kinase C.

Prolactin (PRL) and other trophic factors rapidly activate a nuclear pool(s) of protein kinase C (nPKC) in purified splenocyte nuclei. The PRL also enhanced [2-3H]glycerol incorporation into nuclear mono- and triacylglycerol. An assay was devised which not only probed the ability of the hormone to activate protein kinase C (PKC) but also demonstrated the presence of nuclear substrates. Using this methodology, a biphasic concentration-response curve to PRL was observed. Heterologous species of PRL and various growth factors also activated nPKC. The PRL-induced nPKC stimulation was antagonized by various immunomodulators, G protein-coupling inhibitors, PKC inhibitors, a calmodulin inhibitor, and a peripheral benzodiazepine agonist and antagonist. A monoclonal antibody to PKC, anti-rat PRL antiserum and a monoclonal anti-rat PRL receptor antibody antagonized PRL-induced PKC-dependent nuclear phosphorylation, further implicating nPKC and a PRL receptor-mediated activation process. Nuclear PKC may be a major target for trophic regulation in response to both positive and negative growth signals.

Strategies of immune reconstitution: Effects of lymphokines on murine T cell development in vitro and in vivo

Need for more effective treatment to reconstitute T cell immunity in secondary immunodeficiencies like AIDS prompted an exploration into the roles played by leukocyte products on the ontogeny of murine T lymphocytes in vitro and in vivo. It was observed that mixed lymphokines potently stimulate the proliferation of prothymocytes, immature cortical thymocytes, and mature medullary thymocytes. The effect of the natural, mixed lymphokines could be reproduced in the main by the combination of recombinant interleukin I and II. Mixed lymphokines administered in vivo augmented splenic lymphoproliferative responses in athymic nude mice without T cell marker (Thy 1.2) induction and, in neonatal mice, induced both Thy 1.2 and proliferative responses. Recombinant IL-2 at equivalent dose was less active in nude mice and not active in neonatal mice. The evidence indicates that lymphokines, particularly IL-1 and IL-2 in combination, regulate T cell ontogeny and can act in an endocrine fashion to promote T cell development in T cell-free mice having a functional thymus. Mixed lymphokines may be useful for immune reconstitution in AIDS, however, only if given prior to thymic destruction.

Effects of cytokines on human thymic epithelial cells in culture: IL1 induces thymic epithelial cell proliferation and change in morphology.

The role of thymic epithelium in T cell development has given rise to a number of studies, but less information is available concerning the factors regulating thymic epithelial cells (TEC) themselves. Several cytokines, natural or recombinant, were investigated for their effects on human TEC proliferation. This study presents evidence for the first time that human recombinant interleukin 1 (IL1) and IL1-containing mixed cytokine preparations induced DNA synthesis of TEC as measured in a 48-hr stimulation assay. The effects of IL1 were dose dependent and sustained in time. The following recombinant cytokines, IL2, IL3, IL4, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF alpha), and TNF beta, as well as thymosin fraction 5 and Escherichia coli lipopolysaccharide (LPS), were not found to modify TEC proliferation but IFN-gamma and TNF alpha enhanced the effects of IL1. We also report that IL1 induced a profound change in the morphology of TEC. Our observations suggest that TEC are targets for the action of cytokines and emphasize the important role played by IL1 within the thymus.

Methyl inosine monophosphate: a potential immunotherapeutic for early human immunodeficiency virus (HIV) infection.

MIMP is a new thymomimetic purine under development for immunorestorative therapy. Lymphocytes were obtained from eight patients with acquired immunodeficiency disease (AIDS), eight with symptomatic pre-AIDS (ARC), and 22 normal controls and were stimulated in vitro with phytohemagglutinin (PHA). AIDS patients (mean CD4 counts of 40) showed PHA responses less than 10% of control while ARC patients (mean CD4 counts of 544) showed responses approximately 50% of the control responses. MIMP (0.1, 1, 10 and 100 micrograms/ml) progressively augmented the PHA responses in all these groups. The augmentation of the responses of the leukocytes of AIDS patients while statistically significant was minimal. The augmentation of the responses of ARC patients was significant and their maximal responses approached control levels. The effect of 1 micrograms/ml MIMP was comparable with that observed with indomethacin (10(-6) M) and interleukin-2 (IL2 - 4 units/ml) and was additive with each of these stimulants. In a parallel manner, MIMP restored the suppression of control lymphocytes induced by the immunosuppressive 17 amino acid fragment of the P41 peptide of HIV. In vivo experiments showed that MIMP significantly delayed death in a murine FLV AIDS model at a dose of 1 mg/kg by the oral or parenteral route. MIMP is under preclinical development for early HIV disease to forestall progression to AIDS by attenuating virus-induced immunosuppression.

Thymosin, interleukins, isoprinosine and imuthiol do not reconstitute T-cells in athymic nude mice.

Previous reports suggest that immunotherapy can induce T-cell development in athymic nude mice. Eight-week-old BALB/c nu/nu (athymic nude) mice were treated for 3, 6 and 12 weeks with thymosin fraction 5 (TF-5), buffy coat interleukins (BC-IL), recombinant IL-2 (rIL-2), a combination of TF-5 and BC-IL, isoprinosine or imuthiol. Control animals were treated with sterile saline or were given a syngeneic thymus graft. Spleen weight, cell yields and Thy 1.2+ T-cell markers were monitored and spleen cell proliferative responses to rIL-2, concanavalin A (Con A), Con A + rIL-2, phytohemagglutinin (PHA) and endotoxin (LPS) were assessed. Only mice transplanted with a syngeneic thymus showed progressive increases in both T-cell number and function. Minor changes in proliferative responses were noted at 3 weeks with all treatments; however, no biologically significant reconstitution of T-cell number or function was observed.

A pituitary factor induces thymic epithelial cell proliferation in vitro

Human thymic epithelial cells (TEC) were grown in culture and confirmed to be keratin positive (98-100%) and epidermal growth factor (EGF) responsive. Bovine pituitary extracts (BPE) stimulated the proliferation of TEC. The proliferation of TEC was confirmed by cell counts and radioautography. The BPE was active as measured by tritiated thymidine incorporation in the absence of serum and in the absence of EGF. Individual anterior pituitary hormones (growth hormone, prolactin, ACTH, FSH, LH, TSH) and posterior pituitary hormones (vasopressin and oxytocin) were inactive alone to stimulate TEC proliferation. The effect of EGF but not BPE was blocked by an antibody to EGF suggesting that the active component of BPE is not EGF. Purification of the factor is in progress. The observations suggest that this pituitary-derived factor(s) may regulate thymic function in vivo.

Therapy of secondary T-cell immunodeficiencies with biological substances and drugs

Thymus-dependent (T) lymphocyte defects are common in cancer. Recent advances in the understancling of the regulation of T-cell development by biologicals and drugs now allow the formulation of better strategies of immune reconstitution to correct these defects. Thymic hormone preparations of several types offer one type of approach; however, they are somewhat limited in their reconstitutive ability. Interleukins (IL), particularly IL-1 and IL-2, appear to be complementary to the actions of thymic hormones in promoting T-cell development. Two classes of thymomimetic drugs have been identified and are represented by levamisole and isoprinosine. These drugs mimic by indirect and direct actions, respectively, the actions of thymic hormones. Newer analogs of these compounds have emerged which appear more effective. Also new factors, e.g. pituitary factors, are emerging which may be potent regulators of the immune system and useful in therapy. These agents may now be more effectively integrated with cytodestructive therapy in cancer treatment.