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The effect of histamine (H) and H1-, H2-receptor blocking agents was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood lymphocytes (PBL) from eight healthy subjects on HEP-2 adherent human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of 3H-TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay with a Concanavalin A (Con A) dose of 25 micrograms/ml at 50:1 effector-target cell ratio. Under these conditions, but without Con A, considerable NCMC was not elicited by normal lymphocytes. The presence of histamine and the H2-receptor blocker cimetidine resulted in a significant NCMC to HEp-2 cells. On the contrary, histamine and cimetidine reduced LDCC. The H1-receptor blocker clemastine had no significant effect on either NCMC or LDCC to HEp-2 targets. The possible involvement of H2-receptor bearing cells in the regulation of cytotoxicity to HEp-2 cells is suggested.

We developed a sensitive bioassay system for the determination of tumor necrosis factor-alpha (TNF) using HEp-2 adherent human epipharynx carcinoma cells as targets. TNF from separated human monocytes was triggered by lipopolysaccharide (LPS). In a 24 hr 3H-thymidine incorporation assay, TNF-like activity was seen to reproducibly destroy radiolabeled target cells, i.e., inhibits thymidine incorporation and causes detachment of adherent HEp-2 cells. HEp-2 cells were insensitive to human interleukin-1 (IL-1) and interleukin-2 (IL-2). In contrast, human interferon-alpha and gamma were also cytotoxic for target cells. Monocyte supernatants stimulated by LPS, however, failed to contain detectable amounts of interferons.

Assessment of immune reactivity of patients with disorders of immunopathological origin requires a well-equipped clinical laboratory. The complex mechanism of immune reactions cannot be studied by simple techniques, although the most sophisticated methods do not often prove more reliable than simpler ones. While humoral immune reactivity can be measured by determining serum immunoglobulin levels, the assessment of cellular reactivity requires more complex methods; for this purpose lymphocyte marker studies, mitogen induced blast transformation, LIF production, and skin reactivity proved to be the methods of choice.

Incubation of human peripheral blood mononuclear cells in the presence of 25 mg/l concanavalin A (Con A) or 2 mg/l phytohaemagglutinin (PHA) suppressed their ADCC activity. Thirty-minute incubation with the mitogens resulted in a significant decrease in ADCC activity. The effect was more striking with longer (24 and 48 h) incubation. The suppressive effect of PHA was abolished after 6 days incubation, while no such phenomenon was observed with Con A. Macrophages participating in the ADCC reaction were not influenced by the lectin treatment, though their removal increased the suppressive effect. The lectin-induced suppression of ADCC activity did not correlate with the suppression of Con A-induced blastogenesis. The suppressive effect of lectins on ADCC is not mediated through suppressor cells, but rather represents a direct action of ligands on the (killer) lymphocyte membranes, resulting probably in an altered metabolism, or inhibition of membrane mobility or lymphocyte locomotion.

Con A-induced cytotoxic activity of human lymphocyte subpopulations obtained by cell fractionation procedures was studied in a test system using human epipharynx carcinoma cells (HEp 2) as targets. Only T lymphocytes were cytotoxic, non-T cells exerted no cytotoxic activity, but enhanced the adherence of the tumour cells. Tnon-G lymphocytes (Fc-receptor negative T cells) were more active than TG cells (Fc-receptor-positive T cells) in mediating the Con A-induced cytotoxic reaction.

The effects of cyclosporin A, prostaglandin E1 and indomethacin were studied on lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC activity by human peripheral blood lymphocytes was evaluated by detachment from the monolayer of [3H]thymidine-prelabelled HEp-2 cells in a 24-h assay at 50:1 effector:target cell ratio in the presence of 25 micrograms/ml concanavalin A. Under these conditions, but without concanavalin A, considerable natural cell-mediated cytotoxicity was not elicited although LDCC was significantly augmented in the presence of concanavalin A. Addition of both cyclosporin A (0.1, 1.0 or 10 micrograms/ml) and prostaglandin E1 (10(-8), 10(-7) or 10(-6) M) dose-dependently suppressed LDCC activity. Indomethacin (0.1, 1.0 or 10 micrograms/ml) did not in itself influence LDCC although suppression of LDCC by cyclosporin A, but not prostaglandin E1, was abrogated in the presence of indomethacin. Similar to indomethacin, acetyl salicylic acid also reversed the inhibition of LDCC by cyclosporin A. In parallel experiments, cyclosporin A elicited a more than two-fold increase of prostaglandin E production under LDCC assay conditions as measured by radioimmunoassay. Contrary to LDCC, depression of concanavalin A induced blastogenesis by cyclosporin A was not influenced by indomethacin, suggesting that the inhibition by cyclosporin A of LDCC and concanavalin A-induced blastogenesis proceed via different mechanisms.

Patients with Sjögren's syndrome were treated with vitamin A (100,000 U) daily during a two-week period. The vitamin treatment significantly elevated their ADCC and NK activity. The lymphocyte blast transformation, however was not noticeably changed. After the treatment, the retinyl-ester and retinol level of the plasma significantly increased as did the plasma level of vitamin E. The level of TBA reactive substances (malondialdehyde) in the plasma increased, whilst the glutathione peroxidase and catalase activity of erythrocytes decreased. The activity of the plasma glutathione peroxidase increased, but not significantly.

Interleukin-1 and tumour necrosis factor-alpha activity by E. coli lipopolysaccharide-triggered monocytes was studied in patients with systemic lupus erythematosus in various stages of activity. Monocytes from both groups of SLE patients produced significantly less tumour necrosis factor-alpha activity than those of age and sex matched healthy controls. However, interleukin-1 activity was only significantly reduced in patients with active stage of the disease. These findings indicate further immunoregulatory disturbances in monocyte function concerning SLE.

In this study, earlier observations concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against 3H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.

Interleukin-1 and tumor necrosis factor-alpha activity by E. coli lipopolysaccharide-triggered monocytes were studied in patients with chronic alcoholic liver disease. Monocytes from cirrhotic patients were shown to have a significant reduction in IL-1 and TNF-a activity, compared with that from age- and sex-matched healthy controls. These findings indicate further immunoregulatory disturbances concerning alcoholic liver cirrhosis.

Treatment of patients suffering from systemic lupus erythematosus with vitamin A (100,000 U daily for 2 weeks) resulted in an enhancement of antibody-dependent cell-mediated cytotoxicity, natural killer cell activity and blastogenic response to plant mitogens and interleukin-2 (IL-2).

The basal and stimulated intracellular cyclic AMP (cAMP) levels of peripheral blood mononuclear cells (PBMC) of 16 control subjects and 14 patients with systemic lupus erythematosus (SLE), all fulfilling the ARA criteria, were studied. No significant difference in basal cAMP level was observed between SLE patients and controls. SLE lymphocytes (both active and inactive) elicited a diminished response to aminophylline and prostaglandin E2 (PGE2). No correlation was seen between disease activity and either baseline cAMP levels or response to these stimulators. We suggest an intrinsic (not disease activity-related) impairment of the adenylate cyclase-dependent regulatory mechanism in the PBMC of SLE patients, which may result in a defective IL-2 production and IL-2 dependent biological functions.

The role of OKT4+ and OKT8+ T cell subsets was studied in depressed lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 target cells by peripheral blood mononuclear cells (PBMC) from patients with active systemic lupus erythematosus (SLE). LDCC activity was evaluated by detachment from the monolayer of 3H-TdR-prelabelled HEp-2 cells in a 24 hr assay at 50:1 effector-target cell ratio in the presence of 25 micrograms/ml concanavalin A (Con A). Decreased levels of LDCC were performed by all studied effector cell populations of SLE patients, including both OKT4+ and OKT8+ T cell fractions. LDCC by isolated OKT8+ T cells was superior to that by OKT4+ and unfractionated T lymphocytes from all healthy and SLE subjects. This suggests that the defect of LDCC activity in SLE did not affect the inherently higher LDCC effector activity of OKT8+ to OKT4+ cells. In parallel studies a reduced proliferation of PBMC in response to Con A and failure of OKT8+ T cells to suppress Con A-induced blastogenesis was observed in patients with SLE.

Systemic autoimmune diseases are generally treated with immunosuppressive agents. In some instances immunomodulatory agents have shown promise in the treatment of certain types of autoimmune disorders. The in vitro and/or in vivo effects of some of these agents (glutaurine, ketoconazole, gutimine and its derivative) are demonstrated. Glutaurine exerts moderate immunostimulating activity, but fails to influence the clinical course of systemic lupus erythematosus. Although ketoconazole suppresses immune responses in vitro, it does not influence cellular reactivity of patients in vivo. The immunostimulatory activity of gutimine and its derivative (T 001) have been demonstrated in vitro, and need to be tested also in vivo.

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.

Natural cell-mediated cytotoxicity /NCMC/ was evaluated using human adherent 3H-thymidine-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio in a 24 hr assay. For lectin-dependent cell-mediated cytotoxicity /LDCC/ studies cultures contained also 25/micrograms/ml concanavalin A /Con A/. Peripheral blood mononuclear cells /PBMC/ of nine patients with active systemic lupus erythematosus /SLE/ failed to exert NCMC or LDCC against HEp-2 targets. In contrast, an increased adherence /decreased detachment from the monolayer/ of HEp-2 target cells was observed in the presence of PBMC from SLE patients that was further promoted by the addition of Con A during LDCC assay.

The effect of carrageenan (CGN) was studied on lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of 3H-TdR pre-labelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 micrograms/ml concanavalin A (Con A). Under these conditions but without Con A considerable cytotoxicity was not elicited by peripheral blood mononuclear cells (PBMC). Addition of CGN to the system significantly increased LDCC to HEp-2 targets. Monocyte depletion of effector cells had no major influence to the effect of CGN on LDCC activity. In parallel studies CGN also enhanced the Con A-induced proliferation of PBMC.

Lectin-dependent cell-mediated cytotoxicity of peripheral blood mononuclear cells from patients with uterine cervix carcinoma was evaluated using human adherent 3H-TdR-prelabeled HEp-2 epipharynx carcinoma cells as targets at a 50:1 effector-target cell ratio with 25 micrograms concanavalin A/ml in a 24-h assay. Peripheral blood mononuclear cells from 13 patients with cervical carcinoma in stage IV failed to exert cytotoxicity against HEp-2 targets. In contrast, an increased survival (decreased detachment from the monolayer) of HEp-2 cells was observed in the presence of peripheral blood mononuclear cells from patients, this being significantly enhanced by addition of concanavalin A during the lectin-dependent cell-mediated cytotoxicity assay.

Lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood mononuclear cells (PBMC) from tumour patients was evaluated using human adherent 3H-TdR-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio with 25 micrograms/ml concanavalin A (Con A) in a 24h assay. PBMC of nine patients with metastasizing solid tumour failed to exert cytotoxicity against HEp-2 targets. In contrast, increased survival (decreased detachment from the monolayer) of HEp-2 cells was observed in the presence of PBMC from tumour patients that was significantly enhanced by addition of Con A during LDCC assay.

A cytomorphological analysis of the effect of adult testes on growth and differentiation of grafted fetal testis or ovaries was performed in hamsters. Fetal gonads, taken at 14 days post-coital age, were grafted for 30 days either under the renal capsule or testicular capsule of scrotal or cryptorchid testes of adult hamsters (weight 115 +/- 23 g). Renal grafts were also performed in males castrated 30 days prior to receiving the fetal gonads. Growth and differentiation of the fetal gonads (testis or ovary) was totally inhibited by the scrotal testis. When the cryptorchid testis was the recipient of fetal gonads, inhibition was correlated inversely with the degree of spermatogenic damage elicited in the cryptorchid testis. No inhibition was observed in fetal gonads grafted under the kidney capsule, nor in castrated, normal or cryptorchid animals. As normal growth and differentiation of both testis and ovary occurred when grafted under the kidney capsule, the inhibitory effect of adult gonads seems to be unrelated to plasma testosterone levels in the host, as levels were undetectable in castrated hamsters and reduced drastically in cryptorchid animals. At the same time, the testicular-inhibiting substance in normal animals did not act at a distance, since its effect was restricted to fetal gonads grafted under the testicular capsule. This inhibitory substance may correspond to the spermatogonial chalone, known to be produced by differentiating spermatogenic cells (mainly spermatocytes and round spermatids in the rat and mouse); these chalones prevent spermatogonial proliferation and, consequently, the critical number of spermatogonia needed to enter meiosis is not attained. It is doubtful if the same substance has the ability to differentiate the fetal ovary or if this effect can be ascribed to a more complex situation involving other testicular peptides of paracrine action and/or locally high levels of androgens.

Interleukin-1 and tumour necrosis factor-alpha activities by E. coli lipopolysaccharide-triggered monocytes were studied in patients with chronic alcoholic liver disease. Monocytes from cirrhotic patients were shown to have significantly reduced IL-1 and TNF-a activities, compared with that from age and sex matched healthy controls. These findings indicate further immunoregulatory disturbances concerning alcoholic liver cirrhosis.

The relationship of lectin-dependent cell-mediated cytotoxicity (LDCC) to interleukin-2 (IL-2) production was studied in healthy subjects and in patients with systemic lupus erythematosus (SLE). Profoundly depressed levels of LDCC were elicited by peripheral blood mononuclear cells (PBMC) from nine patients with active SLE in comparison to LDCC from seven controls, and eleven inactive SLE donors, using 3H-TdR-prelabelled adherent HEP-2 cells as targets in a 24 h assay with 25 micrograms/ml Con A. In parallel experiments, no individual correlation was found between LDCC activity and IL-2 production for healthy or SLE subjects. Further, no major differences were detected in IL-2 release when the three groups of donors were compared, a tendency observed at the Con A doses (5 and 25 micrograms/ml) and incubation times (24, 48, and 72 h) used to induce IL-2 production. In additional studies, impaired Con A-induced blastogenesis was noted for PBMC from active SLE patients in comparison to the PBMC from the controls or patients with inactive SLE. While strong individual correlation was obtained between blastogenesis and IL-2 secretion in controls and patients with inactive SLE, no such relationship was found in patients with active SLE. While addition of exogenous IL-2 to the cytotoxicity assay considerably enhanced LDCC by healthy donors it failed to improve LDCC by patients with active SLE. These data suggest that depressed LDCC and Con A-induced blastogenesis of patients with active SLE may not be related to impaired IL-2 production but rather to an inherent dysfunction of the effector lymphocytes, including their unresponsiveness to IL-2.

Treatment with vitamin A (100,000 U daily for 2 weeks) of patients suffering from chronic lymphocytic leukaemia resulted in an enhancement of antibody-dependent cell-mediated cytotoxicity, natural killer cell activity and blastogenic response to plant mitogens.

Lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood mononuclear cells (PBMC) from patients with stage I cancer of the larynx (LC) was evaluated using human adherent 3H-TdR-prelabeled HEp-2 carcinoma cells as targets at 50:1 effector-target ratio with 25 micrograms/ml concanavalin A (Con A) in a 24-hour assay. Under these conditions, but without Con A, no considerable natural cell-mediated cytotoxicity (NCMC) was performed by PBMC either from control or from LC donors. Depressed levels of LDCC, but augmented ADCC to chicken red blood cells were detected in LC patients. Natural killer activity to K562 targets was not different from that of control subjects. In parallel studies, normal Con A-induced blastogenesis and B cell counts, low T, and active T cell counts, as well as high Leu-11a+ cell counts were detected in patients with LC. The relationship between depressed LDCC and low T, and active T cell counts, and enhanced ADCC and high Leu-11a+ cell counts is suggested in stage I LC patients.