Transcriptional analysis of human peripheral blood mononuclear cells after influenza immunization.

Influenza A virus is a major cause of morbidity and mortality worldwide. There is a large knowledge base on the immune response to influenza. However, few studies have focused on global gene expression in immune cells after antigenic challenge. A better understanding of the host immune response is required for the development of more efficient means of prevention and treatment of influenza. In this study, global gene expression in peripheral blood mononuclear cells (PBMCs) after influenza immunization was analyzed. The differential gene expression in antigen-stimulated and non-stimulated PBMCs was determined by cDNA microarrays. To determine whether a specific gene profile was present during a proliferative memory cell response to influenza antigens, gene expression in response to PHA was compared with antigen-stimulated PBMCs. PHA induced the upregulation of 201 genes while influenza virus antigen upregulated more than triple that is 630 genes out of 1700 genes analyzed. Both influenza antigen and PHA commonly upregulated 138 genes. Interferon (IFN)-related genes were induced by influenza but not by PHA. The interferon-gamma induced protein precursor 10 (IP-10) was upregulated 27-fold while the interferon-induced 54 kDa protein exhibited a 13-fold increase. The following gene families were also selectively upregulated by influenza antigens: complement ligands and receptors, T cell activation genes, growth factors, genes related to antigen processing and inflammatory responses. With PHA, the genes TNF-R, CTSG, CD3 delta, C8B, CRF1 and CCR2 had higher expression compared with the viral antigen stimulation. Neutrophil defensins alpha-1 and two C-C chemokines, proteins MIP-1-beta and MIP-4, were among the genes upregulated by both PHA and influenza antigens. The results suggest that interferon-induced genes are one of the main transcriptional targets during the immune response to influenza virus.

Human immunodeficiency virus-1 envelope glycoproteins and anti-CD4 antibodies inhibit interleukin-2-induced Jak/STAT signalling in human CD4 T lymphocytes.

Human immunodeficiency virus (HIV) infection leads to a profound T cell dysfunction well before the clinical onset of acquired immunodeficiency syndrome (AIDS). We have been accumulating evidence that one of the mechanisms responsible for this T cell deficiency may be the dysregulation of signal transduction via the interleukin (IL)-2/IL-2 receptor (R) complex. In CD4 T cells, we have observed previously that viral envelope (env) glycoproteins induce IL-2 unresponsiveness and the down-regulation of the three chains making up the IL-2R (alpha, beta, gamma) in vitro. We have now established further that this disruption of the IL-2/IL-2R system manifests itself in defective signal propagation via the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway in response to IL-2. The treatment of CD4 T cells with HIV env or surface ligation of CD4 with anti-CD4 monoclonal antibodies inhibited the IL-2-induced activation of Jak-1 and Jak-3, as well as their targets, STAT5a and STAT5b. This Jak/STAT deficiency may contribute to the crippling of CD4 T cell responses to a cytokine central to the immune response by HIV.

Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt's lymphoma B cell line BL30/B95-8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt's lymphoma B cells.

IL-4 and IL-13, cytokines with similar biological effects may influence growth and progression of B-cell tumors through regulation of key cell surface molecules important in intercellular communications. In this study, we demonstrate that IL-4 and IL-13 exhibited differential effects on CD23 and CD44 expression and binding to hyaluronan in BL30/B95-8, a Burkitt's lymphoma (BL), and MK3.31, an Epstein-Barr virus transformed normal human B cell line (B-LCL). Studies conducted to understand the molecular mechanisms underlying this differential effect show that IL-4 induced phosphorylation of JAK1, JAK3, and STAT6 in BL30/B95-8 cells and of JAK3 and STAT6 in MK 3.31 cells. In contrast, IL-13 failed to induce the phosphorylation of JAK kinases or STAT6 proteins in these cell lines. The inability of BL30/B95-8 cells to respond to IL-13 was attributed to the loss of expression of IL-13R subunits alpha1 and alpha2, a finding confirmed for a number of other BL cell lines examined.

Unstimulated human CD4 lymphocytes express a cytoplasmic immature form of the common cytokine receptor gamma-chain.

As a component of various cytokine receptors, common cytokine receptor gamma-chain (gamma(c)) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of gamma(c) (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding gamma(c) gene. This apparent paradox was solved by the demonstration that polyclonal anti-gamma(c) Abs detected endoglycosidase-H-sensitive immature forms of gamma(c) (54-58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature gamma(c) is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature gamma(c) is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of gamma(c) is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of gamma(c) may require intracellular interactions with lineage- or subset-specific molecular partners.

Interferon-gamma inhibits CD44-hyaluronan interactions in normal human B lymphocytes.

The interaction of CD44 with its ligand hyaluronan (HA) plays a vital role in lymphopoiesis, lymphocyte homing, T cell activation, and metastasis. This study addresses the effect of cytokines involved in B cell growth on CD44-HA interactions in normal human B cells. Activation of B lymphocytes with LPS, pokeweed mitogen, or anti-IgM antibodies with or without IL-2 or IL-4 failed to induce HA adhesion. Stimulation of B cells with the phorbol ester PMA, however, induced strong HA recognition, which was inhibited by IFN-gamma and to some extent by IL-4. Investigation of the potential molecular mechanism involved revealed that PMA-induced HA adhesion correlated with enhanced expression of CD44-H- and V6-containing isoforms, as determined by flow cytometry, and the differential induction of V4- and V5-containing isoforms, as determined by reverse transcriptase-based polymerase chain reaction analysis. The inhibition of PMA-induced adhesion by IFN-gamma and IL-4 correlated with the downregulation of CD44 H expression and altered usage of exons V4 and V5. However, changes in the electrophoretic mobility of CD44 proteins, as a measure of posttranslational modifications, were not detected in response to PMA and IFN-gamma or PMA and IL-4. These results suggest that the inhibition of PMA-induced HA adhesion by IFN-gamma and IL-4 may influence B cell migration through their ability to downregulate CD44-HA interactions.

Regulation of CD44-hyaluronan interactions in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4.

In this study, we investigated the regulation of CD44-hyaluronan (HA) interactions in a panel of EBV+ Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV transformation of normal human B cells. The results show that among B cell mitogens, phorbol 12-myristate 13-acetate (PMA) alone induced strong HA recognition in EBV+ BL-30/B95-8 cells. Among the cytokines that affect B cell growth and differentiation, IL-4 alone induced HA recognition in BL-30/B95-8 cells. Attempts to delineate the molecular mechanism for this increased HA adhesion in BL-30/B95-8 cells revealed an enhanced expression of CD44 H, isoforms containing V3, V6, and V9 exons, alterations in the splicing pattern of the V4 exon, and the increased electrophoretic mobility of the CD44 H protein. In contrast, the ability to recognize HA was not observed in B-LCL cells stimulated with either PMA or IL-4, even though these cells respond to IL-4, as observed by upregulation of CD23 expression. The molecular pathways that regulate CD44 expression and CD44-mediated HA binding may be selectively inactivated in B-LCL cells. These results may have implications with respect to the generation and spread of B cell tumors.

IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-alpha.

IL-10, an immunoregulatory cytokine produced by T cells and monocytes, inhibits the expression of inflammatory and hemopoietic cytokines as well as its own expression. To evaluate the regulation of IL-10 production by T cells and monocytes, we measured IL-10 levels by ELISA in supernatants of PHA-stimulated PBMC following depletion of either T cells or monocytes. IL-10 production was significantly down-regulated in both T cell- and monocyte-depleted PBMC compared with undepleted PBMC, and IL-10 production could be restored by the addition of monocyte-conditioned medium (supernatant of PHA-stimulated, T cell-depleted PBMC), suggesting that IL-10 production by T cells is regulated by a monokine(s) produced by activated monocytes. To further clarify the monokine(s) responsible for IL-10 induction, we stimulated monocyte-depleted PBMC, purified CD4+, and CD8+ T cells with PHA and measured IL-10 production by ELISA and semiquantitative reverse transcriptase-PCR following monokine(s) addition. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte-depleted PBMC in a dose-dependent and additive manner. Furthermore, anti-IL-6 and anti-IL-12 Abs neutralized the IL-10-inductive effect of monocyte-conditioned medium. Similarly, IL-12 and IL-6 induced IL-10 production by purified CD4+ and CD8+ T cells. With respect to regulation of IL-10 produced by monocytes, TNF-alpha was found to induce IL-10 production by resting as well as by LPS-stimulated purified monocytes/macrophages. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and/or IL-6 can induce the expression of IL-10 by PHA-stimulated T cells, whereas TNF-alpha induces IL-10 production by monocytes. Since IL-10 inhibits the production of IL-6, IL-12, and TNF-alpha, these results may indicate a potential mechanism of negative feedback regulation of the immune response.

Interleukin-6 expression in cord blood of patients with clinical chorioamnionitis.

The objective of this study was to define whether IL-6 is an early marker of infection in the newborn. To correlate the occurrence of clinical chorioamnionitis with the levels of IL-6 expression in neonates, IL-6 was measured in cord plasma by ELISA and in mononuclear cells by reverse transcriptase-PCR before and after mitogenic stimulation. Eight neonates were included in each of the following four groups: elective cesarean section, uncomplicated normal spontaneous vaginal delivery, delivery after prolonged rupture of amniotic membranes with no evidence of chorioamnionitis, and delivery with evidence of chorioamnionitis. All 32 neonates were clinically well after delivery, and all 16 babies with prolonged rupture of membranes or clinical chorioamnionitis had negative blood cultures. Elevated IL-6 levels were found only in neonates born to mothers with chorioamnionitis (119.7 +/- 33.5 pg/mL versus 2.71 +/- 0.59 pg/mL, p < 0.005). Mononuclear cells from five of these neonates expressed no IL-6 mRNA in vivo despite elevated levels of IL-6 in their cord plasma. Cord blood mononuclear cells from healthy term babies were capable of synthesizing IL-6 in vitro in response to stimulation with bacterial lipopolysaccharide. These results suggest that IL-6 levels in cord plasma increased with clinical chorioamnionitis, despite the lack of evidence of infection in the neonates. Therefore, we conclude that, although a high level of IL-6 may be a good marker of chorioamnionitis, it may not be a specific marker of infection in the newborn.

Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines.

Thermal stress induces expression of a family of heat shock proteins which may regulate the synthesis of various cellular genes. We investigated the effect of heat shock on polyadenylation in Epstein-Barr Virus (EBV) negative and EBV transformed human Burkitt's lymphoma (BL) B-cell lines. Incubation of the BL B-cell line P3HR-1, carrying the defective EBV genome [EBV nuclear antigen-2 gene deletion] at 46 degrees C for 15 min increased nuclear poly(A) polymerase (PAP) activity. Thereafter, enzymatic activity declined and at 60 min it was reduced to about 50% of that observed in cells incubated at 37 degrees C. In contrast, no significant increase in PAP activity was observed at 15 min or thereafter in an EBV- BL cell line, ST-486, in response to elevated temperature. Furthermore, no heat shock mediated change in nuclear poly(A)-specific endonuclease activity was observed in either P3HR-1 or ST-486 cells suggesting a specific effect on PAP activity. However, thermal stress dependent increase in c-myc expression was detected only in P3HR-I cells. These results suggest an association between EBV transformation and enhanced expression of c-myc and PAP activity. To further determine the role of EBV, and EBV- BL cell line, BL-30, and BL-30 cells infected in vitro with a wild type strain of EBV, BL-30/B95-8, were investigated. BL-30/B-95-8, unlike the parental BL-30 cells, exhibited c-myc and PAP gene upregulation at 15 min but were downregulated at 60 min following exposure of cells to elevated temperatures. These results suggest that infection of human B-cells with EBV is associated with their ability to respond to thermal stress by increased PAP activity which may stabilize mRNA through enhanced polyadenylation.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients.

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.