Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) anti-genpulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.     

Lack of species specificity in the action of immunosuppressive factors of tumor cells and absence of competition between them and recombinant interleukin-2 and phytohemagglutinin for lymphocyte membrane receptors

We studied some possible mechanisms of action of immunosuppressor factors (ISF) produced by tumor cells on lymphocyte proliferation. ISF of murine tumor cell lines inhibited the mitogen induced proliferation of murine splenocytes as well as human mononuclear blood cells. Normal human mononuclear blood cells or concanavalin A-activated murine spleen cells preincubated with phytohemagglutinin (PHA) or interleukin 2 (IL-2) respectively, were strongly suppressed by ISF in response to these activators. When preincubated with splenocytes or blood cells for 2 h at 4 degrees C following washing, ISF suppressed the lymphocyte proliferation as effectively as when being with cells during all period of cultivation. ISF inhibited mitogen-induced lymphocyte proliferation at low dilutions. There was no competition for lymphocyte membrane receptors between these functionally heterogenic kinds of ISF. Collectively, these results show that ISF acted when being attached to some lymphocyte membrane receptors.     

The effect of a peat-based preparation on mitogen-induced proliferation of thymocytes in non-treated and hydrocortisone-suppressed mice.

The studies were carried out on Balb/c mice (5-6 weeks of age) treated with a peat-based preparation (PBP), administered i.p. once or four times at 24 h intervals at doses of 0.01; 0.1 or 1 mg/kg. Additionally, hydrocortisone was injected i.p. to selected mice at a single dose of 125 mg/kg. The results show that PBP temporarily enhances the proliferative capability of murine thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The effect of PBP depends on the number of subsequent doses, but does not depend on the dose applied. A single PBP administration does not affect the proliferative response of thymocytes to Con A and PHA. A single injection of PBP (doses from 0.01 to 1 mg/kg) does not change the number of thymic cells and weight ratio of this organ. Increased doses of subsequent PBP injections (0.01 and 0.1 mg/kg) do not affect the number of thymocytes, but temporarily increase the weight ratio of the thymus two days after the last injection. Administration of PBP prior to hydrocortisone prevents the suppressive effect of the drug on proliferative response of thymocytes stimulated in vitro with Con A and PHA, at the same time increasing the proliferative response of thymic cells to the two mitogens in relation to the control group (hydrocortisone-free). The effect of a single dose of PBP depends on the dose applied--the weakest preventive effect was observed at a dose of 0.01 mg/kg. An increase in the number of subsequent PBP doses, irrespective of a dose applied, prolongs the protective action of the drug on proliferative activity of thymocytes stimulated in vitro with these mitogenes. Moreover, the results obtained in the studies show that PBP partially prevents the suppressive effect of hydrocortisone, as the number of thymic cells and weight ratio of this organ drastically decreased. PBP accelerates regeneration of the thymus, but this depends on a dose applied and the number of subsequent doses. The result was the strongest and the fastest when PBP was injected four times at a dose of 1 mg/kg. It seems quite likely that the thymic regeneration due to PBP is connected with the effect of this drug on maturation and differentiation of thymic cells.     

Comparative analyses of the effect of triacontanol on photosynthesis,photorespiration and growth of tomato (C3-plant) and maize (C4-plant)

Tomato (C₃-plants) and maize (C₄-plants) were grown in a nutrient solution to which triacontanol was added twice a week. After about 4 weeks the triacontanol treatment caused a significant increase in the dry weight of the tomato plants. Leaf area and dry weight measurements of tomato leaves at different stages of development showed that the largest increase in growth was obtained when triacontanol treatment was initiated before bud formation. In maize, no effect of the triacontanol treatment on dry wieght was observed. Photosynthesis was inhibited by 27% in young leaves from triacontanol-treated tomato plants and 39% in the controls, when the oxygen concentration was raised from 2% to 21%. In maize no change in photosynthesis could be observed, neither after altered oxygen concentration nor after triacontanol treatment. The difference in the response of C₃- and C₄-plants to triacontanol indicates that it regulates processes related to photosynthesis.     

Purification to Homogeneity of Biologically Active Human Interleukin 1

Human interleukin 1 (IL-1) produced by lipopolysaccharide-stimulated monocytes was purified to homogeneity with retention of biological activity. IL-1 was measured by its ability to enhance the proliferative response of thymocytes to phytohemagglutinin. The purification procedure included hydrophobic affinity chromatography on phenyl Sepharose, gel filtration through Ultrogel AcA54 and preparative isoelectric focusing. Both charged species of IL-1, pi 5.1 and 6.8 have a molecular weight of 14,500 as determined by SDS-polyacrylamide gel electrophoresis. The complete purification resulted in a recovery of approximately 0.01% of IL-1 protein and if protection against losses by denaturation and adsorption in the final purification step was provided by bovine serum albumin, approximately 11% of IL-1 activity can be recovered.     

Evaluation of the radioprotective action of mexamine based on the cellularity index of the rat thymus

Tie administration of mexamine leads to emigration of thymus cells levelling the radioprotective effect of the compound as determined by total cellularity of the organ. Processes of thymus cell depletion were additive after the effect of mexamine and ionizing radiation. It was found possible to estimate the radioprotective efficiency of mexamine with regard to thymus tissue cellularity diminution after the administration of the preparation.     

Pineal control of immune status and hematological changes in blood and bone marrow of male squirrels (Funambulus pennanti) during their reproductively active phase

In addition to pineal control of reproduction in seasonal breeders, melatonin is also known to influence various immune parameters. In the present experiment, we assessed the effect of exogenous melatonin treatment on different hematological parameters of peripheral blood and bone marrow cells, together with histological observations of spleen and thymus blastogenic response and stimulation ratio, and hormonal assays (melatonin and testosterone) of Indian palm squirrel (Funambulus pennanti) during their reproductively active phase when endogenous melatonin levels are low. Daily subcutaneous injection of melatonin (25 microg/100 g body mass.) at 17.30-18.00 h to adult male squirrels for 60 consecutive days during May-June significantly increased the lymphocyte count of blood and bone marrow and the blastogenic response/percent stimulation ratio of spleen and thymus. Histological observation showed densely packed thymocytes and splenocytes. During this period, peripheral testosterone level was high and melatonin was low establishing an inverse relationship as noted earlier for this squirrel. In pinealectomized squirrels, decreased total leukocyte count and percent lymphocyte count in peripheral blood and bone marrow, along with a decreased cell density in spleen and thymus was observed histologically. Further, melatonin treatment of pinealectomized squirrels resulted in restoration of the immune parameters in line with a normal control level. We suggest that during the reproductively active period of male Indian palm squirrels the lymphoid organs were sensitive to melatonin; hence, the exogenous melatonin treatment had an immuno-enhancing effect.     

Assessment of leukotriene B4 synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods

Leukotrienes (LT), mainly LTB4, have been shown recently to affect several functions of human lymphocytes in vitro, and they are regarded as putative modulators of the immune response. Although it is recognized that human neutrophils, eosinophils, monocyte-macrophages, and mast cells can generate LTs, the synthesis of 5-lipoxygenase products by lymphocytes is still the subject of a controversy. Human peripheral blood mononuclear leukocytes, nylon wool-purified lymphocytes, CD4+, CD4- T cells, large granular lymphocytes, and various fractions of pure lymphocyte preparations obtained by counter flow centrifugal elutriation were stimulated for 10 min to 24 hr with ionophore A23187, phytohemagglutinin, concanavalin A, or lipopolysaccharide with or without exogenous arachidonic acid (AA); supernatants were analyzed by reverse-phase high performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) methods for the presence of LTB4. Pure human lymphocyte preparations, which were shown to be free of monocytes, did not release any detectable amount of LTB4. Increasing percentage of contaminating monocytes was clearly paralleled by increasing amounts of LTB4. Murine thymocytes, interleukin 2-dependent CTLL2 cytotoxic lymphocytes, EL4 thymoma cells, and human Jurkatt cells were also found to be unable to generate detectable amounts of LTB4 after stimulation with ionophore A23187, phytohemagglutinin, phorbol myristate acetate, recombinant interleukin 1, or interleukin 2 with or without exogenous AA. The addition of increasing numbers of adherence-purified monocytes to Jurkatt cells was followed by increased synthesis of LTB4. In conclusion, the present study indicates that the synthesis of LTB4 by pure human lymphocyte preparations or some human and animal lymphoid cell lines is not detectable by combined HPLC-RIA methods in any of the conditions used.     

Effect of highly purified thymus factor on the cellular and humoral indices of immunity in thymectomized mice]

Effect of homogeneous polypeptide thymus factor of mol weight about 5000 (thymarin-III) on cellular and humoral immune responses of thymectomized adult CBA mice was studied. Thymectomy proved to greatly decrease the number of T-cells in the spleen. Accordingly, the capability of these mice to produce both IgM and IgG antibody-forming cells in response to the thymus-dependent antigen (sheep red blood cells) was significantly depressed. Subcutaneous injections of thymarin-III (1 microgram per g of body weight) for 7 days completely restored the T-cell spleen population and normalized the animals' immune response.     

Accumulation of eosinophils and monocytes in lymphoid organs of chick-embryos. I. Effect of antigenic stimulation.

The effect of antigenic stimulation on the migration pattern of eosinophils and monocytes was studied during the embryonic stage in chickens. On the 13th embryonic day, chickens were injected with sheep red blood cells as antigen into the allantoic cavity and the relative frequency of oxidase positive cells (OPC) was determined as the total number of eosinophils and monocytes in the bursa of Fabricius, spleen, and thymus. Three and five days after the antigenic stimulation, the frequencies of OPC increased in both the spleen and thymus and then decreased to the normal level just before hatching. However, bursal frequencies of OPC were always low in both the cortex and medulla when compared with the controls. These events indicated that eosinophils and monocytes accumulated in the spleen and thymus after the antigenic stimulation. Furthermore, different frequencies of OPC among the embryonic lymphoid organs showed different responses in the migration of eosinophils and monocytes.     

Phytohemagglutinin--a modulator of activity of cytochrome P-450-dependent enzymes of hepatocyte and immunocyte membranes

The phytohemagglutinin effect in vivo on monooxygenase activity depends on the type of the cells under study (thymocytes, splenocytes, macrophages, hepatocytes) and on the period after lectin injection. Variations of monooxygenase activities associated with different forms of cytochrome P-450 are established to have a number of peculiarities in isolated hepatocytes as compared to those in the liver microsome fraction. These differences are supposed to be due to the effect of nonmicrosomal cytochrome P-450. The results obtained are discussed with regard for phytohemagglutinin effect on systems regulating activity of cytochrome P-450-dependent monooxygenases.     

Effect of chronic psychoemotional stress on subpopulation spectrum of T-lymphocytes in immunocompetent organs in male mice

Subpopulation spectrum of T lymphocytes (CD3+, CD4+, CD8+, CD25+, and CD3(+)25+) in thymus, spleen and inguinal lymphatic nodes have been studied in male mice after 20 days of psychoemotional stress produced by social defeats in daily agonistic confrontations. A reduction of total number of cells, of absolute numbers of all researched subpopulations of lymphocytes and % CD3+ cells in thymus of submissive mice was shown in comparison with intact animals. Reduction of total number of splenocytes and absolute numbers of CD4+ and CD8+ lymphocytes has been observed in a spleen of submissive mice. Besides, % CD3+, CD25+, CD4+ and CD25+ cells were increased in these animals in comparison with intact mice. The absolute number of cells with CD8 phenotype was increased in inguinal lymphatic nodes. The data obtained suggest that the chronic psychoemotional stress is accompanied by serious changes of the cellular link of immunity. The effect of chronic emotional social stress on mutual interaction of the central and peripheral links of immunity has been discussed.     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

Impact of treadmill exercise on early apoptotic cells in mouse thymus and spleen

Lymphocyte apoptosis occurs in response to stressors such as thermal injury, trauma, sepsis, and surgery. This study evaluated the effect of a single bout of physical exercise stress on the induction of apoptosis in murine thymocytes and splenic lymphocytes. Female C57BL/6 mice, treadmill exercised at a submaximal intensity (35 m/min, 6% grade) for 90 min or serving as controls (walking on treadmill at 12 m/min, 6% grade, 5 min), were sacrificed 5 min or 120 min after completion of exercise. The percent of apoptotic, necrotic, and viable thymocytes and splenocytes were determined by flow cytometry using annexin V FITC and propidium iodide. There was a significantly higher percent of viable splenocytes in the mice sampled 120 min after cessation of exercise than treadmill control animals (p<0.05). In the thymus, there was a significantly lower percent of apoptotic (p<0.5) and a significantly higher percent of viable (p<0.05) cells in exercised mice sampled at 120 min after exercise relative to controls. Absolute numbers of thymocytes and splenocytes did not differ by exercise treatment condition. Plasma corticosterone levels were elevated immediately after exercise and were negatively correlated with the percent of viable lymphocytes in the spleen. During the time frame sampled, submaximal exercise is associated with a lower % of thymocytes expressing early markers of apoptosis, despite elevated plasma corticosterone levels. Retention of self-reactive, viable thymocytes which would normally be deleted or selective trafficking of apoptotic thymocytes out of the thymus may be involved in the exercise effect. Additional studies are necessary to identify the mechanisms for this shift in proportions of apoptotic and viable cells in lymphoid compartments with exercise.     

Social isolation stress enhanced liver metastasis of murine colon 26-L5 carcinoma cells by suppressing immune responses in mice

We investigated the effect of social isolation stress on the formation of experimental liver metastasis resulted from intraportal vein (i.p.v.) injection of colon 26-L5 carcinoma cells in male Balb/c mice, and elucidated some of the underlying mechanism involving the effects of this stress on cellular immunity. Increases in the colony number and tumor burden were observed in the mice socially isolated before and/or after tumor cell challenge, as compared with the group-housed mice. In addition, exposure of mice to 2 weeks of preisolation resulted in decreases in the thymus weight and number of thymocytes by 35.8% and 40.2%, respectively, in comparison with the controls. Reduced proliferative response of splenocytes to various stimuli and suppressed splenic NK activity, as well as decreased macrophage-mediated cytotoxicity, were also found in the mice exposed to social isolation. Thus, these results suggest that social isolation stress enhances tumor metastasis in part via its suppressive effect on the immune system of the host.     

The role of monocytes in human lymphocyte activation by mitogens.

Studies were performed to determine the role of monocytes in human lymphocyte activation by mitogens. Velocity sedimentation at 1 x G in a new apparatus was utilized to obtain highly purified lymphocyte fractions (LF) nearly free of monocytes (0.02 to 0.4%) and a fraction (MF) enriched for monocytes (64 to 92%). The average peak responses of the lymphocyte fractions to phytohemagglutinin, concanavalin A, and pokeweed mitogen were 19, 10, and 9% of the responses achieved with unfractionated lymphocyte cultures containing approximately 20% monocytes. These changes were not attributable to altered dose requirements. When mitomycin-C-treated MF cells were used to reconstitute LF cultures, it was found that 4% monocytes fully restored the response to phytohemagglutinin whereas 8 to 16% monocytes were required for a normal response to the other mitogens. Higher numbers of MF cells produced supranormal responses, with 35 to 50% monocytes resulting in the optimal stimulation. Allogeneic monocytes were able to fully reconstitute the response of LF, and 2-mercaptoethanol (50 microM) was only slightly effective. In exploring possible mechanisms by which monocytes potentiate the mitogenic activity of lymphocytes, it was found that the supernatants of MF cultures could partially, but not completely, reconstitute LF responses, suggesting that contact with MF may be required for optimal effectiveness. Addition of graded numbers of monocytes to LF altered both the kinetics of the response and the peak level of proliferation. Monocyte depletion also resulted in markedly decreased survival of cultured unstimulated LF. These observations suggest a variety of possible effects of monocytes in potentiating mitogenic responses, including contact-mediated interactions with lymphocytes (possibly to present the mitogen optimally); enhancement of proliferation kinetics and the size of the responding subpopulation, and maintenance of a requisite growth factor(s) in the culture. Small differences in the monocyte content of cultured lymphocyte preparations may thus account for many of the often observed variations in mitogen responsiveness.     

Antilymphocyte antibodies in various human diseases as a factor of reduced functional activity of T-suppressors

A total of 157 sera from adults and children with rheumatoid arthritis, rheumatic fever, myocarditis, neurodermite, bronchial asthma, wound infections, second degree obesity without symptoms of diabetes were examined. 60% of sera contained high concentrations of antibodies possessing cytotoxicity against thymus cells, but not against bone marrow cells. Sera of healthy children and adults contained no cytotoxic antibodies. Sera cytotoxic against mouse thymus cells inhibited the suppressing activity of mouse splenocytes in experiments on syngeneic transfer, reducing the ability of human lymphocytes to form T-RFC. The latter phenomenon is associated with the decline in the number of T-theophyllin-sensitive lymphocytes, known as T-suppressors.     

Immunoregulatory functions of paf-acether. I. Effect of paf-acether on CD4+ cell proliferation

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.     

Splenocytes Seed Bone Marrow of Myeloablated Mice: Implication for Atherosclerosis

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.