Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders.

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.

Evidence for deficiencies in intracerebral cytokine production, adhesion molecule induction, and T cell recruitment in herpes simplex virus type-2 infected mice.

We examined the intracerebral T cell response in mice infected with neurovirulent HSV-2 strains and an avirulent HSV-1. In HSV-2-infected brains, (i) IL-1beta, TNF-alpha and IFN-gamma mRNA expression was low, (ii) ICAM-1 and VCAM-1 were not induced, (iii) few CD4+ or CD8+ cells were detected. By contrast, in HSV-1-infected brains, (i) cytokine mRNA expression was high, (ii) adhesion molecules were strongly expressed, (iii) many T cells were detected. We suggest that deficient T cell extravasation into HSV-2-infected brain regions is caused by negligible ICAM-1 and VCAM-1 expression, which is due to low expression of critical cytokines.

Strain-dependent cytokine profile and susceptibility to oleic acid anilide in a murine model of the toxic oil syndrome.

The toxic oil syndrome (TOS) was caused by the ingestion of an adulterated rapeseed oil containing oleic acid anilide (OAA). It was characterized by lethal symptoms in the acute phase and by symptoms of idiopathic autoimmune diseases in the chronic phase. The pathogenetic mechanisms remain unclear. In a murine model of TOS we demonstrate strain-dependent effects on the immune system after treatment with OAA intraperitoneally. While C57BL/6 (H-2b) mice develop a polyclonal B cell activation without disease symptoms, most A/J (H-2a) mice suffer an acute lethal wasting disease. These differences are reflected in the splenic cytokine gene expression and secretion and in the Ig production. Increased IgE serum levels and reduced TNF-beta mRNA suggest a Th2 cell response in C57BL/6 mice. In A/J mice, splenocytes express IL-1alpha, IL-10, and IFN-gamma mRNA in vivo and secrete high levels of TNF-alpha in vitro. These observations resemble the human condition in TOS with development of either an acute lethal disease or a chronic autoimmune-like disease. As in other chemical-induced reactions genetic susceptibility seems to be important.

CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis.

Interferon regulatory factor-1 (IRF-1) is a member of a family of transcription factors that regulate an array of genes involved in cell growth, differentiation, and death. Analysis of cytokine expression by stimulated CD4+ cells from IRF-1(-/-) and IRF-1(+/+) mice revealed that IRF-1 deficiency resulted in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of naive cell- and Th1-related cytokines. The altered cytokine profiles of IRF-1(-/-) cells could be explained, in part, by a shift in the representation of subsets of CD4+ cells; IRF-1(-/-) mice exhibited a decreased percentage of naive cells (a major source of IL-2) but increased numbers of memory or effector cells (the source of Th2-related cytokines). We analyzed purified, phenotypically matched memory/effector cells from IRF-1(-/-) and IRF-1(+/+) mice and found that the increased Th2:Th1 cytokine ratio was still evident in the IRF-1(-/-) group, thus suggesting that IRF-1 is involved in the polarization of the cytokine repertoire in CD4+ cells. Our data indicate that IRF-1 plays an important role in the maintenance of CD4+ cell subset homeostasis and in the expression of cytokines by naive and memory/effector cells.

Lipopolysaccharide-induced IL-12 expression in the central nervous system and cultured astrocytes and microglia.

We examined whether the cytokine IL-12 could be induced locally in the brain or in glial cell cultures following LPS treatment. In the brain, expression of IL-12 p35 mRNA was constitutive and did not alter following i.p. injection of LPS. In contrast, IL-12 p40 mRNA was only detectable in the brain of mice given two staggered injections of LPS. Dual labeling in situ analysis revealed IL-12 p40 RNA-positive cells scattered throughout the brain parenchyma, with a small number of these cells being identified as astrocytes, while the majority of IL-12 p40 RNA-expressing cells appeared to be microglia. In cultured microglia or astrocytes, LPS and to a much lesser degree IL-1beta, but not IFN-gamma or TNF-alpha, induced the expression of IL-12 p40 mRNA. Numerous glial fibrillary acidic protein-immunopositive cells colabeled for IL-12 p40 RNA; indicating that LPS-stimulated astrocytes expressed IL-12 in vitro. Immunoblot analysis of lysates from LPS-treated astrocytes revealed the presence of multiple species of 40, 43, 75, and 120 kDa containing the IL-12 p40 protein. Finally, secretion of the IL-12 p75 heterodimer was detectable by ELISA from astrocytes treated with LPS plus IFN-gamma, but not with LPS alone. The findings indicate that IL-12 gene expression can be activated in the brain, with the resident glial cells being a prodigious source of this cytokine. The localized production of IL-12 may have a significant impact on the development of cell-mediated immune responses within the central nervous system.

Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice.

The pathogenetic effector functions of hepatitis B virus (HBV)-specific CD4+, Th1 cells were analyzed in two inbred lineages of HBV transgenic mice, one of which overexpresses the HBV large envelope protein rendering the hepatocytes hypersensitive to the cytopathic effects of IFN-gamma, and another that expresses all of the HBV proteins and replicates the virus in the liver. Transfer of HBV envelope-specific Th1 cells resulted in recognition of viral Ag expressed by hepatic nonparenchymal cells, cytokine release, and a transient necroinflammatory liver disease in both lineages. The liver disease was very severe in the IFN-gamma-sensitive lineage, and it was less severe in the lineage that replicates the HBV genome; nonetheless, in this lineage the Th1 cytokines produced by these cells suppressed viral replication in the liver. These results demonstrate that CD4+ T cells with a Th1 functional phenotype can perform pathogenetic and antiviral effector functions in vivo. This suggests that CD4+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection.

Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines.

Epstein-Barr virus (EBV) transformed human B cells proliferate indefinitely in vitro, and it has been proposed that cytokine-mediated autocrine loops contribute to the maintenance of the lymphoblastoid phenotype. We used a novel multiprobe RNase protection assay to quantify cytokine mRNA species expressed by EBV-transformed lymphoblastoid cell lines (LCL), derived either by the transformation of B cells with B95-8 or wild-type EBV or by the in vitro outgrowth of EBV-associated B cell lymphomas to identify cytokines that are commonly expressed in all LCL and thus more likely to be essential for immortalization of B cells. All 16 LCL expressed high levels of tumor necrosis factor (TNF)alpha, TNFbeta, and transforming growth factor (TGF)beta1 mRNA, while interleukin (IL)-10 transcripts were detected in most LCL but at a lower level. Expression of IL-1alpha, IL-1beta, IL-6, IL-12p35, IL-12p40, IL-13 and IFNgamma mRNA was variable among the LCL tested. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, and IL-5 mRNA were undetectable in all LCL. Furthermore, we found that IL-10, TNFalpha, and TNFbeta mRNA were induced in EBV-negative B cell lines after infection with EBV. These data define common versus idiosyncratic patterns of cytokine expression by LCL and, in the former case, such cytokines as TNFalpha, TNFbeta, and IL-10 emerge as strong candidates that are essential for the autocrine regulation of EBV-immortalized B cells.

T cell differentiation and cytokine expression in late life.

Elderly humans are at significant risk with regard to the incidence and severity of many infectious diseases and cancers. Current theory holds that these late-life vulnerabilities arise, in part, through age-related changes in immune function, particularly in the T lymphocyte lineage. Herein, we discuss how such factors as thymic involution and ongoing T cell differentiation in the peripheral tissues contribute to progressive and irreversible shifts in the state of differentiation of the mature T cell pool. We propose that, by late life, these processes yield a T cell compartment with a suboptimal balance of naive and memory T cell subsets, each with altered, subset-specific programs for cytokine gene expression. As such, the T cell compartment in late life may be more prone to immune deficiency or cytokine-mediated dysregulation in response to new or previously encountered pathogens.

Viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease.

One hypothesis for the etiology of central nervous system (CNS) autoimmune disease is that infection by a virus sharing antigenic epitopes with CNS antigens (molecular mimicry) elicits a virus-specific immune response that also recognizes self-epitopes. To address this hypothesis, transgenic mice were generated that express the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus (LCMV) as self in oligodendrocytes. Intraperitoneal infection with LCMV strain Armstrong led to infection of tissues in the periphery but not the CNS, and the virus was cleared within 7-14 d. After clearance, a chronic inflammation of the CNS resulted, accompanied by upregulation of CNS expression of MHC class I and II molecules. A second LCMV infection led to enhanced CNS pathology, characterized by loss of myelin and clinical motor dysfunction. Disease enhancement also occurred after a second infection with unrelated viruses that cross-activated LCMV-specific memory T cells. These findings indicate that chronic CNS autoimmune disease may be induced by infection with a virus sharing epitopes with a protein expressed in oligodendrocytes and this disease may be enhanced by a second infection with the same or an unrelated virus. These results may explain the association of several different viruses with some human autoimmune diseases.

Molecular and cellular biology of dendritic epidermal T cells.

The function and specificity of gamma delta T cells remain enigmatic. Dendritic epidermal T cells (DETC) expressing an invariant gamma delta TCR represent a well established model system to investigate these key issues. Accumulating evidence supports the initial observation that recognition of a keratinocyte self-antigen by DETC proceeds without a requirement for MHC gene products. In addition, recent data have identified bioactive polypeptides expressed by DETC but not by most other T cells. For example, keratinocyte growth factor appears to be exclusively produced by activated DETC and intestinal intraepithelial gamma delta cells. These findings suggest that DETC may recognize antigen in novel ways as well as perform specialized functions complementary to those normally attributed to T cells.

Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.

CD4+ T cell activation and tolerance induction in B cell knockout mice.

B cell knockout mice microMT/microMT were used to examine the requirement for B cell antigen (Ag) presentation in the establishment of CD4+ T cell tolerance. CD4+T cells from microMT mice injected with exogenous protein Ag in adjuvant responded to in vitro challenge by transcription of cytokine mRNA, cytokine secretion, and proliferation. Peripheral tolerance could be established in microMT mice with a single dose of deaggragated protein. This tolerance was manifested by a loss of T cell proliferation and cytokine production (including both T helper cell type 1 [Th1]- and Th2-related cytokines), indicating that B cells are not required for the induction of peripheral T cell tolerance and suggesting that the dual zone tolerance theory is not applicable to all protein Ags and is not mediated through Ag presentation by B cells.

Intervention of CD4+ cell subset shifts and autoimmunity in the BXSB mouse by murine CTLA4Ig.

In the BXSB autoimmune disease-prone mouse strain, male mice develop severe lupus-like symptoms and die early in life (4-6 mo), whereas females do not. We have previously demonstrated that profound phenotypic and functional changes occur with age in CD4+ cells from BXSB males. CD4+ cells from males (4 mo old) were predominantly CD44high, CD45RBlow, and MEL-14low (activated/memory phenotypes), while the reciprocal phenotypes characteristic of naive cells were prevalent in age-matched females and young adult males (2 mo old). CD4+ cells from older males proliferated less and produced less IL-2 and IFN-gamma than cells from either females or young males in response to immobilized anti-CD3 mAb. We tested the effect of CTLA4Ig treatment on the progression of disease in BXSB males. CD4+ cells from CTLA4Ig-treated mice at 4 mo of age were predominantly CD44low, CD45RBhigh, and MEL-14high phenotypes that were identical with those observed in CD4+ cells from young (3-mo-old) females. In contrast, control male mice treated with IgG2a accumulated the CD4+ memory phenotype. CD4+ cells from 4-mo-old male CTLA4Ig-treated mice proliferated and produced IL-2 at levels similar to those of cells from females in response to immobilized anti-CD3 mAb. Furthermore, in contrast to IgG2a-treated mice, female and CTLA4Ig-treated male mice at 4 mo of age produced no anti-chromatin Abs. Three of four male mice injected with CTLA4Ig until 6 mo of age appeared healthy at 8 mo of age, whereas all five of IgG2a-treated control males died by 6 mo of age. These 8-mo-old CTLA4Ig-treated males showed variable resistance to autoimmunity as well as function and phenotype marker expression, and a less striking glomerulonephritis than 4-mo-old untreated males. The results of this study demonstrate that the rampant T cell activation and T cell dysfunction that occur in male BXSB mice by 4 mo of age are abrogated by blocking the CTLA4-dependent costimulatory signal(s). They also show that treatment with CTLA4Ig can suppress the pathogenesis of disease and increase longevity.

Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes.

It is widely believed that viral clearance is mediated principally by the destruction of infected cells by CTLs. In this report, we use a transgenic mouse model of HBV replication to demonstrate that this assumption may not be true for all viruses. We find that adoptively transferred virus-specific CTLs can abolish HBV gene expression and replication in the liver without killing the hepatocytes. This antiviral function is mediated by IFN gamma and TNF alpha secreted by the CTL or by the antigen-nonspecific macrophages and T cells that they activate following antigen recognition. These cytokines activate two independent virocidal pathways: the first pathway eliminates HBV nucleocapsid particles and their cargo of replicating viral genomes, while the second pathway destabilizes the viral RNA. Intracellular viral inactivation mechanisms such as these could greatly amplify the protective effects of the immune response, while failure of such mechanisms could lead to viral persistence or to the death of the host.

In vivo tolerance induction and associated cytokine production by subsets of murine CD4+ T cells.

Male BXSB mice develop lupus-like disease and die early in life (4 to 5 mo) whereas female mice do not. Others have demonstrated that CD4+ cells from male mice support B cell resistance to tolerance induction to human gamma-globulin (HGG). In this study, male and female mice tolerized at 2 mo of age with deaggregated HGG and subsequently immunized with HGG in comparison with mice immunized only were tested for anti-HGG Ab responses. CD4+ cells from draining lymph nodes of these mice were tested in culture for proliferation and production of cytokine mRNA and protein in response to HGG plus APC. Tolerized male but not female mice produced anti-HGG Abs of both the IgG1 and IgG2a isotypes. HGG-stimulated CD4+ cells from immunized male and female mice that were not tolerized produced IL-2, IL-4, IL-5, IFN-gamma, and TNF-beta mRNA as well as IL-2 and IL-4 protein, whereas tolerized, immunized mice of both sexes failed to proliferate or produce either IL-2 or IL-4 or express any cytokine mRNA in response to HGG in vitro. A resistance in tolerance induction in male mice, as determined by anti-HGG Abs, was also observed at 3 mo of age. Although a resistance to tolerance was also seen in terms of proliferation in the 3-mo-old males, production of IL-2 or IL-4 protein was still not observed. Thus, all T cell subsets identified by cytokine expression profiles were tolerized not only from females but also from males, of which the latter appeared to show some resistance to tolerance induction.

The murine autologous mixed lymphocyte response: distribution of stimulator cells.

Previous studies have shown that neonatal, but not adult, murine thymic T cells proliferate when co-cultured with syngeneic, adult splenic B cells. Evidence suggests that expansion of such self-reactive T cells precedes their clonal deletion or functional inactivation (anergy). This mechanism may be of particular significance for establishing peripheral (extrathymic) tolerance. In order to assess the autostimulatory capacity of B cells present in a variety of lymphoid tissues, neonatal T cells were cultured with Peyer's patch and peritoneal cavity cells. The results indicate that B cells in these tissues readily induce T cell proliferation. Evidence suggests that the B-1 B cell subpopulation is not obligatory for this process. Self-reactive T cells were evident in the spleen of young mice, substantiating a role for B cells in maintaining peripheral tolerance. The results suggest that B cells involved in this process are distributed in a variety of lymphoid organs.

Alteration of intracerebral cytokine production in mice infected with herpes simplex virus types 1 and 2.

Previously we reported that a lethal strain of herpes simplex virus type 2 (HSV-2) infects the brain following ocular inoculation of mice. We now demonstrate that HSV-2 mediates an unusual intracellular sequestering of class II major histocompatibility complex (MHC) antigens. With use of an RNase protection assay, we observed a selective inhibition of IFN-gamma and IL-6 gene transcription in brains of mice infected with HSV-2. It is likely that the inhibition of cytokine gene expression was mediated through a failure to activate CD4+ lymphocytes. These data suggest that the infecting herpesvirus can influence the profile of intracerebrally produced cytokines, which in turn may determine the outcome of the infection.

Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo.

To investigate the mechanism by which viruses are cleared from neurons in the central nervous system, we have utilized a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (OBLV60). After intranasal inoculation, OBLV60 grew preferentially in the olfactory bulbs of BALB/c mice. Using in situ hybridization, we found that viral RNA localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. Virus was cleared rapidly from the olfactory bulb between 5 and 11 days. Athymic nude mice failed to eliminate the virus, demonstrating a requirement for T lymphocytes. Immunosuppression of normal mice with cyclophosphamide also prevented clearance. Both CD4+ and CD8+ T-cell subsets were important, as depletion of either of these subsets delayed viral clearance. Gliosis and infiltrates of CD4+ and CD8+ cells were detected by immunohistochemical analysis at 6 days. The role of cytokines in clearance was investigated by using an RNase protection assay for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and gamma interferon (IFN-gamma). In immunocompetent mice there was upregulation of RNA for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma at the time of clearance. Nude mice had comparable increases in these cytokine messages, with the exception of IFN-gamma. Induction of major histocompatibility complex class I (MHC-I) molecules on cells in infected brains was demonstrated by immunohistochemical analyses in normal and nude mice, suggesting that IFN-gamma may not be necessary for induction of MHC-I on neural cells in vivo.