Effects of weak laser radiation (632.8 nm) on isolated mouse immune cells

The dose dependence of in vitro effects of low-intensity radiation of a He-Ne laser (632.8 nm, 0.2 mW/cm²) on the functional activity of peritoneal macrophages and lymphocytes of mouse spleen was studied. The exposure of isolated cells was varied from 5 to 180 s. If the exposure did not exceed 60 s, stimulation of secretory activity was observed: increased production of interleukin 2, interferon γ, and interleukin 6 in lymphocytes; increased production of tumor necrosis factor α, nitric oxide, and interleukin 6 in macrophages; and enhanced activity of natural killer cells. A longer exposure (up to 180 s) either had no effect on the synthesis of certain cytokines (interleukin 2 in lymphocytes and interleukin 6 in macrophages) or inhibited it, which was expressed in decreased production of interleukin 6 and interferon γ in lymphocytes and nitric oxide in macrophages, as well as in suppression of the activity of natural killer cells. Conversely, the production of interleukin 3 decreased after a short-term exposure but increased after 180-s irradiation. The high sensitivity of cells to extremely weak laser light also manifested itself as a considerable increase in expression of the inducible heat shock protein 70; this effect was observed at all doses studied, including the 5-s exposure. In contrast, expression of the heat shock protein 90 slightly decreased after irradiation of cells with laser light.     

Role of interleukin 1 and interleukin 2 in rat and mouse arthritis models

The production of interleukin 1 (IL 1) and interleukin 2 (IL 2) by macrophages and lymphocytes from three animal models commonly used for rheumatoid arthritis, viz. adjuvant-induced and type II collagen-induced rat arthritis, and MRL/1 murine arthritis was studied. Although the peritoneal macrophages from adjuvant-arthritic rats in culture produced increased amounts of prostaglandin E2 (PGE2) and lower levels of IL 1 than the control group, cells from collagen-arthritic rats released normal levels of PGE2, but increased amounts of IL 1. After activation with lipopolysaccharides, the IL 1 production by macrophages from all groups was comparable. Addition of indomethacin did not significantly change the IL 1 production in any of these groups. In the absence of any exogenous mitogen, IL 2 production by the lymphocytes of adjuvant-arthritic rats was low, but could be restored to the normal levels when phytohemagglutinin A (PHA) or concanavalin A (Con A) was added. The lymphocytes from collagen-arthritic rats were capable of producing IL 2 without the need of any T cell mitogen. The lymphocytes from MRL/1 mice seemed to lack the functionality in terms of IL 2 production. The macrophagic IL 1 production in these animals was normal. Our data suggest that the type II collagen arthritis model may closely resemble human rheumatoid arthritis in which IL 1 and IL 2 production by the mononuclear cells is significantly enhanced.     

Effects of sleep deprivation on human immune functions

The effect of 40 h of wakefulness on a variety of immunological parameters in the peripheral blood from 10 normal male subjects was studied. Sleep deprivation led to enhanced nocturnal plasma interleukin 1-like and interleukin 2-like activities. The rise in nocturnal response of lymphocytes to pokeweed mitogen stimulation during a normal 24 h sleep-wake cycle was delayed by sleep deprivation, but the response to the phytohemagglutinin mitogen was unaffected. With resumed nocturnal sleep, there was a prolonged decline in natural killer cell activity (measured as spontaneous cytolytic activity for human tumor cells) and return of an increased response to pokeweed mitogen. The altered patterns in immune functions occurred independently of the cortisol circadian rhythm, which remained unchanged.     

Effect of triacontanol on numbers and functions of cells involved in inflammatory responses.

A preparation of a triacontanol-containing compound was studied for its effect on cells involved in the inflammatory response. C57BL/6 mice were injected intraperitoneally with various concentrations of this compound and investigated for total body weight, wet weight of thymus tissue, number of thymus cells and splenocytes, interleukin 1 production of spleen monocytes, and response of splenocytes to the T cell mitogen, phytohemagglutinin. Mice treated with the triacontanol preparation exhibited decreased total body weight, 24% reduction in thymus weights, 39% decrease in the number of thymus cells, and 21% depression in total splenocytes. Splenic monocytes of these animals produced a significantly reduced amount of interleukin 1 and splenocytes had a significantly depressed response to phytohemagglutinin. It is concluded that triacontanol has an inhibitory effect on at least some of the cells responsible for inflammation.     

Continuous endotoxin infusion suppresses rat spleen cell production of cytokines.

Endotoxin, i.e., lipopolysaccharides, was continuously infused into rats at a nonlethal dose by means of an implanted osmotic pump for up to 2 weeks. The pump was connected to the jugular vein by a polyethylene catheter. Administration of endotoxin via the pump compromised the ability of spleen cells to produce the lymphokines interleukin 1 and tumor necrosis factor after stimulation in vitro with endotoxin. In addition, the ability of the spleen cells to produce alpha/beta-interferon in response to endotoxin in vitro was also examined, as was the capability of the spleen cells to produce gamma-interferon following stimulation with concanavalin A. Suppression of the expected interleukin 1 and tumor necrosis factor production by spleen cells from rats continuously infused with endotoxin was observed. There was also a moderate effect on interferon production, but this was much less. These results provide further findings indicating the unresponsiveness of spleen cells to lipopolysaccharides, as well as to a nonspecific plant mitogen, following continuous infusion of endotoxin into rats via an implanted osmotic pump. Additional studies are needed to determine the mechanisms involved in such suppression.     

Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson "single-hit" relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations.     

Studies on immunomodulatory properties of isoniazid. II. Effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression

The effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression by phytohemagglutinin-stimulated human T cells was investigated. High concentrations of the drug decreased interleukin 2 production while low doses (10(-5)-10(-6) M) produced a slight increase in interleukin 2 production. Isoniazid did not affect the expression of interleukin 2-receptors on the surface of T cells, except a slight decrease in cells exposed to high levels of the drug.     

Absolute requirement for adherent cells in the production of human interleukin 2 (IL2)

Activated T lymphocytes appear to require a growth factor, interleukin 2 (IL2), for continued proliferation. It has been hypothesized that T lymphocytes serving an amplifier function produce IL2 in response to an activating signal such as antigen or mitogen; in addition, the producer lymphocytes receive a second signal from macrophages. The present experiments demonstrated that human IL2 production in culture is totally depleted by exhaustive removal of adherent cells and can be completely restored by replacement of the adherent cells.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.     

Modulation of lymphocyte proliferation and immunoglobulin synthesis by interferon-gamma and "type I" interferons

Interferon (IFN)-alpha and IFN-beta ("type I" IFNs), but not IFN-gamma reduced phytohemagglutinin- or pokeweed mitogen (PWM)-induced proliferation in cultures of human mononuclear leukocytes. Proliferation induced by specific antigens (tuberculin PPD or tetanus toxoid) or by exogenous interleukin 2 (IL-2) was strongly inhibited by type I IFNs and, to a lesser extent, by IFN-gamma as well. Inhibition of proliferation in mitogen-stimulated cultures was not due to a reduced production of IL-2 or to an inhibition of IL-2 receptor expression. Type I IFNs inhibited immunoglobulin (Ig) production in PWM-stimulated unseparated mononuclear cells, whereas IFN-gamma enhanced Ig production in such cultures. In cultures of purified B cells type I IFNs caused a stimulation of Ig production and this B-cell differentiation factor (BCDF)-like activity of IFNs was synergistically enhanced in the presence of IL-2. IFN-gamma produced less BCDF-like activity than type I IFNs. These results show that in some instances type I IFNs can be more potent in affecting functions of cells of the immune system than IFN-gamma.     

Requirement for extracellular calcium or magnesium in mitogen-induced activation of human peripheral blood lymphocytes

The importance of calcium in lymphocyte activation is well recognized, but the levels of extracellular ionized free calcium (Ca++) necessary for lymphocyte proliferation via various pathways have not been investigated in detail. We studied the ability of a lectin mitogen (PHA) and a calcium ionophore (ionomycin) to induce interleukin 2 receptors, interleukin 2 (IL2) production, and proliferation over various concentrations of extracellular Ca++. Reducing the Ca++ levels from the normal 200 microM to 10 microM in PHA-stimulated cultures partially inhibited IL2 receptor expression, IL2 production, and subsequent proliferation. At 1 microM Ca++, both IL2 activity and proliferation were eliminated, but partial IL2 receptor expression was still observed. Ionomycin did not induce any of these events in cultures where the extracellular Ca++ concentration was below 100 microM. Restoring calcium in the medium resulted in normal levels of IL2 receptor expression, IL2 activity, and proliferation when PBL were stimulated with either mitogen. Exogenous magnesium partially restored these events in PHA-stimulated cultures, but had no effect when ionomycin was used as the mitogen. These data indicate that stimulation by ionomycin is much more dependent upon the levels of extracellular Ca++ than is PHA. Extracellular calcium also appears to be necessary subsequent to IL2 receptor acquisition, since the latter was seen without IL2 activity or proliferation at very low extracellular Ca++, and IL2 failed to restore the proliferative response under these conditions. The data also suggest that PHA, but not ionomycin, can activate lymphocytes via a magnesium-dependent pathway, or that PHA has a lower specificity for divalent cation cofactors.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.     

VLDL modulates the cytokine secretion profile to a proinflammatory pattern.

Human very-low-density lipoprotein (VLDL) inhibits DNA synthesis in lymphocytes activated by the nonspecific mitogen concanavalin A (Con A). We studied the effects of VLDL on lymphocyte activation (IL-2 receptor expression), cell cycle progression, and production of IL-2 and of IL-4 (a proinflammatory and an anti-inflammatory interleukin, respectively) to understand why an atherogenic lipoprotein inhibits cell proliferation. After 48 h of stimulation with the mitogen, VLDL decreased the population of cells bearing IL-2 receptor and the population of T-cells that progress through the cell cycle, increasing the population of T-cells in G(0)/G(1). Cells cultured in the presence of Con A and VLDL produced higher levels of IL-2 and lower levels of IL-4 than cells cultured without VLDL. These results suggest that VLDL inhibits lymphocyte proliferation by reducing IL-2 receptor and enhancing the levels of IL-2. Probably, one atherogenic effect of VLDL is to modulate the cytokine secretion profile of lymphocytes to a predominantly proinflammatory response.     

Bovine interleukin 2: production kinetics in normal and in vivo antigen-primed cattle

Conditions influencing production kinetics of bovine interleukin 2 (IL-2), viz. cell concentration, mitogen and its concentration, length of incubation, nutrient medium and in vivo antigen-priming were investigated. Peripheral blood mononuclear cells (PBL) of outbred cattle of different age groups showed considerable variation in their ability to secrete IL-2 which possibly reflects their immune competence. Of the cultures initiated with PBL, 5 x 10(6) cells/ml cultured in serum free Iscove's medium and stimulated with 5 micrograms Con A/ml for 24 hr produced maximal amount of IL-2 activity. In vivo antigen-priming of bovine lymphocytes with the live attenuated rinderpest virus revealed that IL-2 production was not affected by rinderpest virus but the in vivo antigen-priming possibly resulted in concomitant production of suppressor factor(s) which suppressed the already produced IL-2. The implications of this factor(s) in relation to regulation of immune responses in the disease process are discussed.     

The inhibitory effects of K+ channel-blocking agents on T lymphocyte proliferation and lymphokine production are "nonspecific"

The effect of K+ channel-blocking agents, tetraethylammonium (TEA) and 4-aminopyridine (4AP), on the responses of cloned murine helper and cytolytic T lymphocytes stimulated with mitogen, anti-T cell receptor monoclonal antibody, or interleukin 2 was examined. The addition of TEA and 4AP reduced [3H]thymidine incorporation and lymphokine production to levels observed in unstimulated cells. However, thymidine incorporation by the tumor cell lines P-815 and SP2/0, which replicate autonomously, also was inhibited by these drugs. Treatment of cloned murine helper T lymphocyte, L2, with TEA appeared to inhibit uptake of [3H]thymidine and [3H]phenylalanine after stimulation with interleukin 2. These results suggest that the inhibitory effects of the K+ channel-blocking agents TEA and 4AP may not be specific for the sequence of events that are initiated by activation of T lymphocytes through the antigen receptor. Instead, the observed inhibitory effects by these agents may result from inhibition of transport of thymidine, amino acids, and other essential metabolites across the cell membrane.     

The effect of x-rays on the precursors of antibody forming cells (B cells) as measured with the in vitro limiting dilution assay

The effect of X-irradiation upon murine antibody-forming cell precursors (B cells) was established in cultures of spleen cells stimulated with the B cell mitogen lipopolysaccharide (LPS). At day 5 and 7 the numbers of IgM- and IgG2-secreting cells were determined in cultures of irradiated and nonirradiated spleen cells. From these numbers a Do of 0.6-1.2 Gy for the IgM, and of 0.9-2.1 Gy for the IgG2 response was calculated. Similar Do values were obtained under limiting dilution culture conditions. In the limiting dilution assay the effect of irradiation upon the size of the IgM-producing clones could also be determined. It was found that irradiation reduced the number of LPS-reactive B cells without affecting the size of the clones produced by the surviving cells.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll‐like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF‐κB‐α) and mitogen‐activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin‐6, chemokine (C‐X‐C motif) ligand 1 (CXCL1) and chemokine (C‐C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2‐deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF‐κB‐α and c‐Jun N‐terminal kinase mitogen‐activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2‐deficient cells,. Inhibitor assay revealed that NF‐κB and MAPKs are essential for B. fragilis‐induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.     

Murine leukemic cells inhibit IL 2 production by susceptible syngeneic splenocytes

BALB/c mice fail to mount significant cell-mediated immunity against the syngeneic virally induced leukemia MCDV-12, and die approximately 10 days after tumor inoculation. Our studies in vitro demonstrated that BALB/c splenocyte and irradiated MCDV-12 cell co-culture led to reduced alloreactivity, including depressed interleukin 2 (IL 2) production. Tumor-induced immune suppression was genetically restricted, antigen nonspecific, and alleviated in part by exogenous IL 2 administration in vitro. Furthermore, mitogen stimulation of IL 2 production, not requiring self or alloantigen recognition, was unaffected by MCDV-12 exposure. These results indicate that tumor cells may escape from immunosurveillance by reducing IL 2 production in the immediate tumor vicinity, and suggest a potential role for major histocompatibility complex antigens in the immunosuppression mechanism.