Alcohol consumption is associated with alterations in macrophage responses to interferon-gamma and infection by Salmonella typhimurium.

Abuse of ethanol (EtOH) by human beings and administration of EtOH to experimental animals has been shown to be associated with a suppression of the immune system. Consumption of EtOH has also been associated with an increased incidence and severity of infections of human beings and experimental animals, which has been attributed to the immunosuppression associated with EtOH consumption. It has been shown that EtOH also affects the function of macrophages (MØ), which are important effector cells in the innate and adaptive immune responses to infectious agents. The present studies were designed to investigate the effects of EtOH on MØ function with an animal model of EtOH consumption. The experiments reported in this paper were done with inflammatory MØ and were designed to determine the effects of EtOH on the ability of inflammatory MØ to respond to interferon-gamma (IFN-gamma) to control the intracellular growth of Salmonella typhimurium, as well as the production of proinflammatory cytokines and nitric oxide. The ability of MØ from EtOH-fed mice to respond to bacterial endotoxin (lipopolysaccharide (LPS)) and IFN-gamma was also evaluated. MØ isolated from EtOH-fed mice did not respond as well to IFN-gamma as MØ isolated from control mice as measured by control of S. typhimurium, as well as tumor necrosis factor (TNF) and nitric oxide production. Interleukin (IL)-6 production was not affected. Activation of MØ from EtOH-fed mice with LPS and IFN-gamma produced levels of nitric oxide and TNF only slightly less than the levels seen in MØ from control mice, but a significant decrease in IL-6 was seen when MØ from EtOH-fed mice were stimulated with this combination. Flow cytometric analyses showed that IFN-gamma receptor expression was not affected by EtOH. Together the data presented in this paper show that consumption of EtOH is associated with changes in inflammatory MØ responses to IFN-gamma.

Regulation of IL-13 production by histamine in cloned murine T helper type 2 cells.

Histamine affects the balance of T helper type 1 (Th1) and T helper type 2 (Th2) cytokines by shifting cytokine production from a Th1 to a Th2 pattern. Interleukin-13 (IL-13) is an important autacoid mediator that has been implicated in the development of allergic disease. This study was designed to investigate the mechanisms of regulation of IL-13 by histamine in Th2 cells. D10.G4.1 cells, a murine Th2 cell line, were treated with histamine (10(-8)-10(-4) M) and then activated with PMA (phorbol 12 myristate 13-acetate) plus ionomycin or alphaCD3. Levels of IL-13 production were then measured by enzyme-linked immunosorbent assay (ELISA) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Cells were pretreated with histamine receptor antagonists pyrilamine, ranitidine, cimetidine and thioperamide to determine the involvement of histamine receptors. Cells were also pretreated with protein kinase A (PKA) inhibitors N-[2-(methylaminoethyl)]-5-isoquinoline-sulfonamide (H-8) and Rp-diastereomer of adenosine cyclic 3'5'-phosphorothionate (Rp-cAMPS), and Janus kinase-signal transducer and activator of transcription (Jak-STAT) inhibitor tyrphostin AG490 prior to the addition of histamine. H-8 is an inhibitor of the catalytic subunit of PKA while Rp-cAMPS is an inhibitor of the regulatory subunit of PKA. Tyrphostin is an inhibitor of Jak2, Jak3, STATI, STAT3 and STAT5. Finally, cells were pretreated with IL-12, a monokine known to repress STAT6 DNA binding. We found that histamine dose-dependently enhanced IL-13 secretion and mRNA levels in Th2 cells via H1 and H2 receptors. Pretreatment of cells with H-8, Rp-cAMPS and tyrphostin prevented histamine-induced secretion and transcription of IL-13. Likewise, pretreatment of Th2 cells with IL-12 also reversed histamine's effects on IL-13 secretion from stimulatory to inhibitory. These observations suggest a role for PKA and the Jak-STAT pathway in histamine-mediated elevation of IL-13 secretion and transcription.

Histamine utilizes JAK-STAT pathway in regulating cytokine production.

Histamine shifts TH1/TH2 cytokine balance from TH1 to TH2 cytokines. The phosphorylation of STAT factors and their translocation to nucleus are important steps in the regulation of TH1/TH2 cytokine balance. This study was designed to investigate the effects of histamine on Janus kinases-signal transducers and activators of transcription (JAK-STAT) pathway. The splenocytes were treated with histamine in the presence or absence of JAK-STAT inhibitor, tyrphostin, activated with IFNgamma for 30 min, and phosphorylated STAT1 was detected by immunoblotting. We found that histamine up-regulated the phosphorylation of STATI and tyrphostin prevented this phosphorylation. We then studied the effects of tyrphostin on histamine-mediated inhibition of IFNgamma production and histamine-mediated stimulation of IL-5 and IL-10 production. Tyrphostin dose-dependently reversed the effects of histamine on IFNgamma, IL-5 and IL-10 production, as evident by ELISA. These observations suggest that histamine regulated JAK-STAT signal transduction, which is involved in cytokine secretion.

The effects of histamine on interferon gamma production are dependent on the stimulatory signals.

Histamine regulates the immune response by enhancing TH2 cytokine production and by inhibiting TH1 cytokine production. We assessed the mechanisms of histamine's action on helper T cell subsets by evaluating the role of protein kinase A (PKA) in the histamine-mediated effects on IFN gamma production. The splenocytes and TH1 murine cloned cells (pGL10) were pretreated with histamine at a concentration range of 10(-8)-10(-5) M for 1 h and then were activated with anti-CD3, PHA, PMA + ionomycin, or ionomycin for 24 h. The levels of IFN gamma were measured in the supernatants by ELISA. The inhibitory effects of histamine were the most prominent in anti-CD3-stimulated splenocytes (61%). The effects of histamine on IFN gamma production from TH1 cells depended on the mode of cell activation. The activation of cells with anti-CD3 resulted in 27% inhibition of IFN gamma production whereas the activation with ionomycin produced 70% suppression. The inhibitory effects of histamine were completely reversed by cimetidine in a dose-dependent manner in both TH1 cells and in splenocytes. PKA played a role in the inhibition of IFN gamma by histamine when the cells were activated via TCR, and the PKA inhibitors Rp-cAMPS (10(-5) M) and H8 (10(-5) M) reversed the inhibitory effects of histamine on IFN gamma production. However, when the cells were stimulated with ionomycin, the PKA inhibitors did not affect histamine-mediated suppression of IFN gamma production.

Regulation of interleukin-10 secretion by histamine in TH2 cells and splenocytes.

Interleukin-10 is a potent suppressive factor that down-regulates cellular immune response via inhibition of the production of TH1 cytokines. Histamine shifts the TH1/TH2 balance from TH1 to TH2 cytokines making the effects of histamine on IL-10 secretion an important factor in this switch. This study was designed to assess the role of histamine in the regulation of IL-10 production and the involvement of PKA and STAT factors in this process. TH2 cells (D10.G4.1) and AKR/j splenocytes were pretreated with histamine at a concentration range of 10(-8)-10(-5) M for 1 h and then activated with PMA + ionomycin or anti-CD3 for 24 h. The supernatants were collected and tested for IL-10 content by ELISA. Histamine stimulated IL-10 production in TH2 cells in a dose-dependent manner that was reversed by both H1- and H2-receptor antagonists and by PKA inhibitors H8 and Rp-cAMPS. Tyrphostin also reversed the stimulation of IL-10 secretion by histamine, indicating that STAT factors were involved in this process. The up-regulation of IL-10 production by histamine in splenocytes was accompanied by inhibitory effects of histamine on IFN gamma production. The pretreatment of splenocytes with histamine in the presence of anti-IL-10 abrogated histamine-mediated inhibition of IFN gamma production suggesting that the effects of histamine on IFN gamma secretion were regulated by IL-10 in multi-cell system.

Investigation of K+ channel expression in human peripheral lymphocytes of healthy donors by means of flow cytometry.

Evaluation of different types of K+ channel expression was performed in resting and PHA (phytohemagglutinine)-activated human peripheral lymphocytes (HPL) of healthy donors by means of flow cytometry. In resting peripheral lymphocytes, the application of kaliotoxin (a selective blocker for voltage-dependent K+ (K(V)) channels), K(V) resulted in pronounced depolarization of lymphocyte membrane potential, with further promotion in the presence of thapsigargin (compound discharging Ca(i) from endoplasmic reticulum). In activated HPL, the expression of various types of K+ channels was estimated utilizing cell-cycle analysis data. In contrast to the resting cells, kaliotoxin-induced depolarization of membrane potential in PHA-activated lymphocytes of the G0/G1 phase was not enhanced by thapsigargin and in PHA-activated lymphocytes of the S and G2/M phases we were able to observe repolarization of membrane potential after kaliotoxin-induced depolarization of membrane potential. Substitution of kaliotoxin for charybdotoxin (a non-selective drug blocking both K(V) and K(Ca) channels) abrogated the above effects in PHA-activated lymphocytes. Thus, K(V) channels are active in both resting and activated HPLs and K(Ca) channel expression occurs with cell-cycle progress on PHA-induced activation of peripheral lymphocytes.

Immunoregulatory effects of N9-benzyl- and N7-benzyl-8-bromoguanines.

In this study we investigated the effects of two guanine derivatives, 9-benzyl- (I) and 7-benzyl-8-bromoguanines (II) on the proliferation of human T-cell leukemia and T-cell lymphoma, normal human peripheral blood mononuclear cells (PBMC), and mouse Th1 (pGL10) and Th2 (D10.G4.1) clones. We also assessed their effects on cytokine production (IL-3, IL-10 and IFN-gamma) in PBMC, T-cell lymphoma, HUT78 (IL-2), and murine Th1 (IL-2) and Th2 (IL-4 and IL-5) clones. These compounds were synthesize as analog of known inhibitors of purine nucleoside phosphorylase (PNP) 8-amino-9-benzylguanine. These compounds suppressed proliferation of human leukemia MOLT-4 cells, human cutaneous lymphoma HUT78 cells and normal PMBC. Compound II was a significantly more potent inhibitor than compound I. Exogenous recombinant human IL-2 reversed the anti-proliferative effects of both compounds on HUT78 cells. These compounds had low toxicity to human EBV-transformed B-lymphocytes. Both compounds suppressed the production of IL-2 by activated human HUT78 cells, IFN-gamma by PBMC and did not affect IL-3 and IL-10 production in PBMC. Compound I inhibited anti-CD3-activated IL-2 secretion from the murine Th1 clone. The murine Th2 clone was less sensitive to both compounds as compared with Thl. The production of IL-4 and IL-5 by this clone was not suppressed. Thus, it has been shown that not only 9-substituted guanines but also their 7-isomers selectively inhibit T-cell functions and both selectively inhibit Th1-related cytokines secretion.

Chronic hepatitis C: T-helper1/T-helper2 imbalance could cause virus persistence in peripheral blood.

Interferon gamma (IFN gamma) and interleukin 4, 10 and 12 (IL-4, -10, -12) production was measured in whole peripheral blood (WPB) and peripheral blood mononuclear cells (PBMC) of 10 chronic hepatitis C (CHC) patients. The level of IFN gamma in supernatants in mitogen-activated WPB was lower than in healthy donors. IL-10 served as a possible downregulative factor for IFN gamma, since its spontaneous IL-10 production was enhanced in CHC. Neutralization of IL-10 partly restored IFN gamma response in CHC patients. Recombinant IL-12 (rIL-12) also enhanced IFN gamma of CHC patients, but IL-12 production was decreased in CHC. Thus, IFN gamma production deficiency in CHC patients is secondary to blockage by high levels of IL-10-impaired IL-12 production.

Interferon gamma production in whole peripheral blood culture in acute hepatitis B.

Interferon gamma (IFNg) production in whole peripheral blood (WPB) and mononuclear (MN) cell culture in acute hepatitis B (AHB) was compared. IFNg production was induced by phytogem agglutinin and measured in the cell supernatants of 14 AHB patients in the course of the disease. There were some up-regulating factors of IFNg production that probably operated in WPB culture: the presence of autoerythrocytes as well as the low content of monocytes. Autoserum regulated IFNg production in a stage-dependent way: it decreased IFNg activity at the bilirubin peak in hepatitis B infection, but not in convalescence. In contrast, we did not find a serum blocking effect in the corresponding stage of acute hepatitis A. The nature of this serum blocking factor in AHB is unclear.

[Immune response and the dynamics of the cytolysis syndrome in viral hepatitis A and B]. / Immunnyi otvet i dinamika sindroma tsitoliza pri virusnom gepatitie A i B.

Forty-nine patients with acute viral hepatitis A and B were examined for the relationship of the blood content of individual lymphocyte subpopulations at the height of the disease to subsequent duration of the cytolytic syndrome (high activity of ALT). The duration of the cytolytic syndrome associated with viral hepatitis A was reversely related to the content of B lymphocytes and when associated with viral hepatitis B to the content of T cells--immunoregulator precursors (autorosette-forming cells). In both patterns of viral hepatitis, the duration of the cytolytic syndrome did not depend on the rate of lymphocyte sensitization to the inducer antigens (HAVAg, kHBsAg). Unlike viral hepatitis A, the duration of the cytolytic syndrome in viral hepatitis B was directly related to the rate of lymphocyte sensitization to hepatic lipoprotein.

[Characteristics of immune response regulation in viral hepatitis A and B]. / Osobennosti reguliatsii immunnogo otveta pri virusnykh gepatitakh A i B.

In 272 patients with virus hepatitis A and B the content of theophylline-sensitive lymphocytes (T-suppressors) and theophylline-resistant lymphocytes (T-helpers) in the peripheral blood was determined. Differences in the content of T-suppressors in cases of acute virus hepatitis A and B with an equal degree of severity were revealed: at the peak of hepatitis A infection in the mild form of the disease the number of the cells was decreased, while at the peak of hepatitis B infection an increase in their number was observed in the mild and moderate forms of the disease and a decrease, in the severe form of the disease. In chronic persistent hepatitis a decrease in the content of T-suppressors and an increase in the content of T-helpers were observed, and in chronic active hepatitis (at the period of remission) and increase in the T-helpers occurred. Changes in the content of the cells of both types are not characteristic of HBsAg carriership.

[Changes in the count of highly avid, thermostable blood T-cells in viral hepatitis and its outcome]. / Izmenenie soderzhaniia vysokoavidnykh, termostabil'nykh T-kletok krovi pri virusnykh gepatitakh i ikh iskhodakh.

The determination of the content of thermostable T-lymphocytes in the peripheral blood in 184 patients with acute virus hepatitis, as well as at the stage of the termination of the disease, has revealed the increased number of cells belonging to this subpopulation in all the groups under study. At the acute stage of the disease a rise in the number of thermostable T-cells is directly related to the severity of the process. Of different variants of the termination of hepatitis B, the highest content of thermostable T-cells is observed in chronic active hepatitis and chronic HBsAg carriership, while in chronic persistent hepatitis the content of thermostable T-cells is considerably lower.