PHA-induced Lymphocyte Proliferation Research Articles

Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol).

Recent studies have suggested that vitamin D may have other important biologic activities in addition to its well-characterized role in the maintenance of calcium homeostasis. Discovery of cytosolic receptors for vitamin D in human peripheral blood monocytes and lectin-stimulated lymphocytes prompted us to study the effects of 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, upon phytohemagglutinin (PHA)-induced lymphocyte blast transformation. We have found that calcitriol is a potent inhibitor of PHA-induced lymphocyte proliferation, achieving 70% inhibition of tritiated thymidine incorporation after 72 h in culture. Furthermore, calcitriol suppressed interleukin-2 (IL-2) production by PHA-stimulated peripheral blood mononuclear cells in a concentration-dependent fashion. Lastly, the suppressive effect of calcitriol on cellular proliferation was partially reversed by the addition of saturating amounts of purified IL-2. We conclude that calcitriol is a potent inhibitor of PHA-induced lymphocyte blast transformation and that this effect is mediated, in part, through suppression of IL-2 production. Thus, calcitriol appears to possess immunoregulatory properties that have been unappreciated heretofore.

Biochemical events associated with lymphocyte activation in aging: K + transport and sensitivity of the Na +K + pump to digoxine

The present investigation was directed toward answering the question of whether some age-related changes of membrane dependent triggering mechanisms during lymphocyte activation could account for the depressed T cell response to mitogens in aging. For this purpose, the K + movements were analyzed in PHA-stimulated peripheral blood lymphocytes (PHA-PBL) from old humans (O) compared to adult (A). Indeed, plasma membrane Na +, K +, ATPase activation plays an essential role in cell proliferation and results from direct interaction between the loaded mitogen receptor and the enzyme. No difference could be found in the magnitude and the timing of the PHA-induced increase of K + fluxes between PHA-PBL from O and A despite a higher K + inflow in unstimulated but 20-hour preincubated PBL from O. Further experiments showed that the lectin-induced triggering mechanism of cation transport resulted from digoxine (DGX: a glycosid cardiotonic) sensitive ATPase. Moreover, whereas PBL from O exhibited a decreased PHA-induced DNA synthesis, DGX depressed the thymidine incorporation by 72-hour cultured PHA-PBL within the same inhibitory dose-related pattern in both O and A. We conclude that the triggering mechanism of Na +, K +-ATPase induced by PHA occures adequately in early stimulated PBL from old subjects. In addition, digoxine sensitive structures work freely during PHA-induced lymphocyte proliferation in aging, thereby supporting further arguments for adequate Na +, K +-ATPase activity.

A study on endocrine disorder in patients with chronic renal failure. I. Synthesis and biological activity of human gastrin I.

A heptadecapeptide amide, H-Pyr-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2, corresponding to the entire amino acid sequence of human gastric I, was synthesized by the solution method. This peptide inhibited lactate dehydrogenase activity by 87.6% at a concentration of 300 pg/ml. This peptide was tested for suppression of PHA-induced lymphocyte proliferation, but showed no significant activity.

In vitro and in vivo effects of diethylstilbestrol and estramustine phosphate (estracyt®) on the mitogen responsiveness of human peripheral blood lymphocytes

The responsiveness to diethylstilbestrol (DES) and estramustinephosphate (EMP) of human peripheral blood lymphocytes to T-cell mitogens has been investigated in vitro and in vivo. EMP demonstrated potent inhibition of both Con A- and PHA-induced lymphocyte proliferation in vitro, while it had no detectable effects when given to patients with cancer of the prostate. DES reduced the response to Con A in vitro, but had only marginal effects on PHA-induced mitogen response. In contrast, the response to Con A was unaltered, while the response to PHA was significantly diminished after DES therapy in patients with prostatic cancer. This effect, however, was only seen when high doses of DES not included in conventional regimen were given. The proliferative response to T-cell mitogens in patients with prostatic cancer was not affected by serum source in the assay, indicating the absence of humoral factors able to inhibit mitogen response in these patients.

Influence of anti-inflammatory drugs on the interaction of lymphocytes and macrophages.

The helper function of macrophages in lymphocyte stimulation is well known, but there are indications that macrophages may also exert a suppressor effect on lymphocytes. This effect might be due to prostaglandins secreted by the macrophages. In order to test this hypothesis anti-inflammatory drugs, some of which are known inhibitors of prostaglandin synthesis, were added to a series of phytohaemagglutinin (PHA)-stimulated lymphocyte cultures containing different proportions of macrophages and lymphocytes. The experiments showed that high concentrations of all drugs were inhibitory. Moderate concentrations of some of the PG-synthesis-inhibiting drugs (like indomethacin and mefenamic acid), however, appeared to have a stimulatory effect. The stimulation was more pronounced in cultures containing a high proportion of macrophages. These results support the assumption that macrophages release prostaglandins, which suppress PHA-induced lymphocyte proliferation.

High immune responsiveness in a family with multiple paraproteinaemia and autoimmune thyroid disease.

Immune responsiveness was investigated in a family comprising 12 first- and second-degree relatives, one of whom had kappa-myelomatosis, one IgA-lambda paraproteinaemia, two Graves' disease and a further two thyroid antibodies without disease. Relatives by marriage served as controls. Parameters of immune capacity studied were the humoral and cellular immune response to haemocyanin of Helix pomatia (HPH), dinitrochlorobenzene (DNCB) skin reactivity and in vitro lymphocyte proliferation capacity to phytohaemagglutinin (PHA). Mean antibody titres to HPH were higher in the family than in the control group in all main Ig classes and IgG subclasses after primary and secondary immunization, and the difference was statistically significant for IgG, IgG2 and IgG4 titres. This could not have been predicted from the (normal) serum Ig levels in this family. In vitro lymphocyte proliferation capacity to HPH after primary and secondary immunization was also significantly increased. DNCB skin reactivity also tended to be high in the family, whereas PHA-induced lymphocyte proliferation was normal. These findings support the idea that myelomatosis clusters in families with immune dysregulation.

PGI 2 and PGD 2 effects on cyclic amp and human T-cell mitogenesis

The effects of PGI 2, PGD 2, PGE 2, and PGF 2α on human peripheral blood lymphocytes (HPBL) cyclic AMP levels and their response to a T cell mitogen (PHA) were investigated. It was determined that although PGI 2, PGD 2, and PGE 2 all elevate intracellular levels of cyclic AMP in HPBL, only PGD 2 and PGE 2 exert potent suppression of PHA-induced lymphocyte proliferation. It is suggested that the instability of PGI 2 in aqueous solution is at least partially responsible for its relative inability to maintain elevated levels of cyclic AMP and concomitant suppression of mitogenesis. PGD 2 was found to exert an effect very similar to PGE 2, both in terms of cyclic AMP elevation and suppression of T cell mitogenesis.

Prolonged impairment of cellular immunity in children with intrauterine growth retardation

Cellular immunity was studied in 17 newborn infants, in eight children aged 1 to 5 years with intrauterine growth retardation, and in age-matched control subjects. At birth T and B peripheral blood lymphocytes were decreased, and delayed cutaneous hypersensitivity to phytohemagglutinin was diminished. In vitro PHA-induced lymphocyte proliferation was similar to that in control subjects but was greater than in healthy adults. In later childhood the numbers of T lymphocytes were normal, but their proliferative capacity was significantly reduced and cutaneous hypersensitivity was minimal or absent. Prolonged impairment of cellular immunity in these children may explain their increased susceptibility to infection and inadequate response to immunization, and predispose to the development of allergic, autoimmune, and neoplastic disease.

The alveolar macrophage: Its capacity to act as an accessory cell in mitogen-stimulated proliferation of guinea pig lymphocytes

The capacity of the alveolar macrophage to act as an accessory cell in PHA-induced lymphocyte proliferation was investigated and compared with that of the peritoneal and peritoneal exudate macrophages in guinea pigs. When lymph node cells were co-cultured with autologous lung cells recovered by airway lavage, the proliferative response to PHA was greatly enhanced over that of lymph node cells alone. In the presence of peritoneal cells or peritoneal exudate (glycogen-induced) cells, the PHA response was intermediate between that of lymph node cells alone and lymph node cells cultured with lung cells. Experiments using purified macrophages (≥98%) as accessory cells demonstrated that the difference observed between lung and peritoneal accessory cells was due to differences in macrophage function. Furthermore, when lymph node cells were cultured in the upper chamber of a double-chambered Marbrook apparatus, PHA-induced proliferation was enhanced only when lung and not peritoneal macrophages were present in the lower chamber. Additional experiments showed that this difference (1) was not an artifact of the thymidine incorporation assay to measure proliferation; (2) was not affected by changing the macrophage-lymphocyte ratio; and (3) was not simply a trephocytic or growth promoting effect of macrophages which could be replaced by 2-mercaptoethanol. These findings show that macrophages from different sources differ in their abilities to act as accessory cells in PHA-induced lymphocyte proliferation. Alveolar macrophages appear to have an enhanced capacity compared to unstimulated and stimulated peritoneal macrophages in this function. At least part of this difference may be due to a difference in the elaboration of soluble factor (s) by macrophages.

Inhibition of Proliferation of Lymphoma Cells and T Lymphocytes by Suppressor Cells from Spleens of Tumor-Bearing Mice

We have recently demonstrated suppressor cells in spleens of mice bearing tumors induced by Moloney murine sarcoma virus (MSV) which were non-T cells and inhibited phytohemagglutinin Molney (PHA)-induced DNA synthesis of syngeneic normal spleen cells. From the present study, the suppressor cells appeared to be macrophages since they were radioresistant, inactivated by carrageenan, and removed by adherence columns and an iron/magnet technique. We have also found that suppressor cells were still fully active when added 16 hr after the mitogen, thus indicating that early mitogen-induced changes were not the target of suppressive action. It appeared that suppressor cells inhibited metabolic events related to the initiation of DNA synthesis and that they had a selective effect on proliferation-dependent lymphocyte effector functions. PHA-induced cytotoxic reactivity which in our system is largely independent of DNA synthesis was not depressed but actually enhanced in MSV spleens. Cytotoxicity of MSV spleen cells against syngeneic lymphoma cells was unaffected by suppressor cells whereas lymphocytes stimulation by mitomycin C-treated syngeneic lymphoma cellls was inhibited. MSV spleen cells also inhibited DNA synthesis of cultured murine lymphoma cells. This function was only slightly diminished after treatment with anti-omicron serum plus guinea pig complement. Furthermore, spleen cells from MSV tumor-bearing nude mice were as effective as spleen cells from their heterozygous littermates, thus suggesting that T lymphocytes are not the main effector cells of inhibition of lymphoma cell DNA synthesis. The inhibitor cells were radioresistnant, inactivated by carrageenan, and removed by adherence columns and the iron/magnet technique. These data strongly suggest that the inihibitor cells of lymphoma cell DNA synthesis are macrophages and that they belong to the same group of cells as the suppressor cells of PHA-induced lymphocyte proliferation.