Susceptibility to cytomegalovirus infection may be dependent on the cytokine response to the virus.

BACKGROUND: Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in an immunocompromised host. Pulmonary infection with CMV results in an inflammatory response, which includes the local production of cytokines. Cytokine production stimulated by CMV infection serves to activate a series of immunologic responses involved in viral clearance. Previous work has demonstrated that different mouse strains express variable sensitivity to CMV infection. METHODS: Using mouse strains that express sensitive (BALB/cj) and resistant (C57BL/6) CMV phenotypes, we asked whether the differences in susceptibility to infection were caused by differences in pulmonary cytokine production after intraperitoneal infection with CMV. RESULTS: C57 mice demonstrated a higher total bronchoalveolar lavage (BAL) and BAL lymphocyte count at 3 and 7 days after intraperitoneal infection compared with BALB mice. There were no differences in BAL cytokine production; however, we were able to demonstrate differences in CMV DNA load in the lungs of BALB mice compared with that of C57 mice. In addition, there appeared to be increased whole-lung production of the TH2 cytokine IL-10 in the BALB mice versus the C57 mice. CONCLUSIONS: This observation suggests that the genetic susceptibility to CMV infection may, in part, be regulated by differences in cytokines production within the local environment.

Immediate early gene 2 of human cytomegalovirus increases interleukin 2 receptor-alpha gene expression.

Previously, we demonstrated that the cytomegalovirus (CMV) immediate early 2 (IE2) gene product upregulates interleukin-2 receptor-alpha (IL-2R alpha) gene expression. To further define this effect, we used a series of IL-2R alpha promoter truncations to identify that the primary site of CMV IE2 activity was in the region between -281 and -273 of the IL-2R alpha promoter, an area without known transcription factor activity. Deletion of known transcription factor enhancer elements resulted in a similar decrease in IE2-driven promoter activation. This unique sequence from the IL-2R alpha promoter was shown to drive a minimal promoter in the presence of IE2. These studies identify a new transcription factor binding site in the IL-2R alpha promoter, which may be specifically responsive to the CMV IE2 gene product. These studies also suggest that one mechanism whereby CMV infection may result in organ transplant rejection is through increased IL-2R alpha expression.

Asbestos stimulation triggers differential cytokine release from human monocytes and alveolar macrophages.

Inhalation of asbestos fibers results in a variety of lung diseases, including pulmonary fibrosis. Various animal models have demonstrated the importance of cytokines in the pathogenesis of pulmonary fibrosis. Alveolar macrophages from patients exposed to asbestos spontaneously release increased amounts of cytokines. The purpose of these studies was to determine whether asbestos directly stimulates cytokine release from human alveolar macrophages after in vitro exposure. We demonstrate that, although asbestos triggers cytokine release from blood monocytes, normal alveolar macrophages do not respond to asbestos stimulation with cytokine release. However, normal alveolar macrophages are activated by asbestos particles, in vitro, as determined by the upregulation of mRNAs for cytokines, and activation of the p38 kinase, which has been shown to be important in the translation of cytokine message into protein. These studies demonstrate that asbestos stimulates both normal blood monocytes and normal alveolar macrophages, but that there is a block in translation of cytokine mRNAs in the macrophages.

Psychiatric disorders and survival after lung transplantation.

The 30 patients who underwent lung transplantation between 1990 and 1996 were included in this study, and data were analyzed to find predictors of 1-year survival posttransplantation. All patients were followed throughout the posttransplantation period. Fifteen patients had a pretransplantation diagnosis of an anxiety and/or depressive disorders. Of the 30 patients transplanted, 19 survived 12 months or more, and 11 died less than 12 months posttransplantation. The > 12-month survival group had a mean age of 45.2 years at transplantation, compared with a mean age of 43.0 years in the < 12-month group (NS). The mean Psychosocial Assessment of Candidates for Transplant score and premorbid history of smoking did not differ between the groups. The > 12-month survival group had more psychiatric illness pretransplantation than the < 12-month survival group (56% vs. 27%, P < 0.05). The recipients with a psychiatric history (N = 15) were more likely to survive 1 year posttransplantation than the recipients without a psychiatric history (80% vs. 47%, P < 0.05) and were not significantly different from the recipients without a psychiatric history in terms of episodes of rejection, bronchiolitis obliterans, or noncompliance with treatment. Depression and anxiety are treatable disorders that occur frequently in patients with end-stage lung disease, and a premorbid history of either did not predict a worse outcome posttransplantation in this study of lung transplantation recipients.

Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes.

Alveolar macrophages play an important role in host defense and in other types of inflammatory processes in the lung. These cells exhibit many alterations in function compared with their precursor cells, blood monocytes. To evaluate a potential mechanism for these differences in function, we evaluated expression of protein kinase C (PKC) isoforms. We found an increase in Ca2+-dependent PKC isoforms in monocytes compared with alveolar macrophages. We also found differential expression of the Ca2+-independent isoforms in alveolar macrophages compared with monocytes. One consequence of the activation of PKC can be increased expression of mitogen-activated protein (MAP) kinase pathways. Therefore, we also evaluated activation of the MAP kinase extracellular signal-regulated kinase (ERK) 2 by the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA activated ERK2 kinase in both alveolar macrophages and monocytes; however, monocytes consistently showed a significantly greater activation of ERK2 kinase by PMA compared with alveolar macrophages. Another known consequence of the activation of PKC and subsequent activation of ERK kinase is activation of the transcription factor activator protein-1 (AP-1). We evaluated the activation of AP-1 by PMA in both monocytes and macrophages. We found very little detectable activation of AP-1, as assessed in a gel shift assay, in alveolar macrophages, whereas monocytes showed a substantial activation of AP-1 by PMA. These studies show that the differential expression of PKC isoforms in alveolar macrophages and blood monocytes is associated with important functional alterations in the cells.

Cytomegalovirus modulates transcription factors necessary for the activation of the tumor necrosis factor-alpha promoter.

Several studies have demonstrated that cytomegalovirus (CMV) infection increases expression of the tumor necrosis factor (TNF) gene. This effect is mediated, in part, by an effect of the CMV immediate early 1 (IE1) gene product on the TNF promoter. To further analyze these interactions, we used plasmids with TNF promoter truncations to determine the site within the promoter where the CMV IE1 gene product mediates its effect. The site was localized to a 40-base pair segment that contains a cAMP response element (CREB). Deletion of the cAMP response element increased basal promoter activation but had little effect on IE1-induced activation. Additional studies demonstrated that the cAMP element is flanked 5' by a PU.1 site and 3' by an NF-kappa B site, both of which increase expression of the TNF promoter. These sequences demonstrated IE1 responsiveness. We next determined the relevance of these observations for normal human cells by infecting human alveolar macrophages with CMV. In these studies we evaluated expression of NF-kappa B, PU.1 and CREB by gel shift assay at immediate early times after infection. We found that CMV infection increased the binding activity of NF-kappa B and PU.1 and decreased the binding activity of CREB. CMV infection also increased expression of the TNF gene in alveolar macrophages. These observations suggest that CMV increases TNF gene expression, in part, by altering the binding activity of transcription factors that regulate gene expression.

Cytomegalovirus modulates interleukin-6 gene expression.

Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent studies have suggested that interleukin (IL)-6 may be used to detect both infection and rejection after lung transplantation. In addition, IL-6 may play a role in the development of bronchiolitis obliterans after transplantation. Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined whether CMV induces IL-6 gene expression. We demonstrated that CMV infection increased both IL-6 protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the CMV immediate early 1 gene product increased expression of the IL-6 promoter. This effect of the CMV immediate early 1 gene product was dependent upon the presence of specific transcription factor binding sites in the IL-6 promoter. These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on IL-6.

Influenza A pneumonitis following treatment of acute cardiac allograft rejection with murine monoclonal anti-CD3 antibody (OKT3).

A 51-year-old man developed fever, cough, and dyspnea 5 days after completing murine monoclonal anti-CD3 antibody (OKT3) treatment for acute cardiac allograft rejection. Samples of BAL fluid grew influenza A virus. Progressive pulmonary infiltrates, respiratory compromise, and hypoxia developed, and the patient ultimately required 5 days of mechanical ventilation. Treatment with amantadine hydrochloride and ribavirin was prescribed, and the patient was discharged after 19 days. Influenza A virus has not been an important pathogen in cardiac transplant recipients. However, this is the first reported case of influenza A pneumonitis complicating anti-T lymphocyte therapy for cardiac allograft rejection. In comparison with our patient, two previously reported cases of influenza A infection in cardiac transplant patients have been less severe. The virulence of our patient's, life-threatening infection appears to be secondary to impairment of T lymphocyte-mediated immunity by OKT3. The role of therapeutic and even prophylactic amantadine therapy in this clinical setting has yet to be determined.

Mycobacterium tuberculosis in lung transplant recipients.

The diagnosis and treatment of Mycobacterium tuberculosis, although difficult in normal hosts, are even more complex in transplant recipients. As a result of the use of immunosuppressive agents, transplant recipients are not only predisposed to primary tuberculous infections, but they are also uniquely at risk for reactivation of latent infection acquired prior to transplantation or transmitted via the donor organ. The diagnosis of pulmonary tuberculosis can be even more elusive in the setting of lung transplantation where other pulmonary complications can make diagnosis difficult. Here we report a case of a patient who died of disseminated M. tuberculosis 12 wk after lung transplantation, and we review tuberculous infections in lung transplant recipients.

Silica increases tumor necrosis factor (TNF) production, in part, by upregulating the TNF promoter.

Silica causes release of tumor necrosis factor (TNF) from mononuclear phagocytes. One hypothesis is that silica increases TNF production, in part, by upregulating the TNF gene. To evaluate this hypothesis, THP-1 cells (a myelomonocytic cell line) were exposed to various amounts of silica and then the TNF gene transcription was evaluated. In this study silica caused a dose-dependent increase in TNF mRNA and the peak response occurred at 3 h following stimulation. A transient transfection assay also showed that silica upregulated expression of a TNF CAT construct in THP-1 cells. Furthermore, a nuclear run-on assay demonstrated that silica particles induce increased TNF gene transcription in exposed cells. THP-1 cells cultured for various periods of time in the presence of silica released TNF into the cell supernatants. These studies show that silica can upregulate the TNF gene, which results in the release of TNF protein from the cells.

Dexamethasone inhibits IL-1 and TNF activity in human lung fibroblasts without affecting IL-1 or TNF receptors.

Interleukin (IL-1) and tumor necrosis factor (TNF) activate human lung fibroblasts through interactions with specific receptors. One effect of this interaction of IL-1 and TNF with fibroblasts is an increased production of the cytokines, IL-6 and IL-8. Dexamethasone blocks the induction of IL-6 and IL-8 by IL-1 or TNF. In these studies, we determined whether dexamethasone interferes with the upregulation of IL-6 and IL-8 by downregulating expression of the IL-1 or TNF receptor genes. Confluent lung fibroblasts were treated with medium alone (control) or medium with dexamethasone (10(-6) M). Dexamethasone did not decrease the binding of IL-1 and TNF to their receptors, nor did it decrease amounts of IL-1 or TNF receptor RNA. Both IL-1 and TNF increased release of IL-6 and IL-8 from the cells in a dose-dependent manner and dexamethasone inhibited this effect. Dexamethasone also inhibited the induction of IL-6 and IL-8 RNA by IL-1 and TNF. The studies show that dexamethasone does not block the effects of IL-1 or TNF on fibroblasts by decreasing expression of IL-1 or TNF receptors.

Regulation of expression of the IL-1 receptor antagonist (IL-1ra) gene by products of the human cytomegalovirus immediate early genes.

Human cytomegalovirus (HCMV), which is a major cause of morbidity and mortality in immunosuppressed patients, can itself alter immune function. It has previously been shown that HCMV immediate early (IE) gene products regulate the IL-1 beta promoter. The purpose of these studies was to determine whether HCMV IE gene products regulate expression of the IL-1 receptor antagonist (IL-1ra) gene. THP-1 cells, a myelomonocytic cell line, were transfected with a plasmid containing one or more of the HCMV IE genes downstream of the HCMV major immediate early promoter, or with a control plasmid. IL-1 beta and IL-1ra protein secretion was evaluated by ELISA, and expression of the mRNA for the cytokines was examined by means of Northern blot analysis. The HCMV IE1+2 gene products were found to increase expression of the mRNA for both IL-1 beta and IL-1ra; however, only the IL-1ra protein was released in increased amounts. The individual HCMV IE gene products had different effects on expression of the IL-1ra gene; the HCMV IE1 gene product down-regulated expression of the IL-1ra gene, whereas the IE2 gene product up-regulated expression of the IL-1ra gene. Thus, HCMV IE gene products can either up-regulate or down-regulate expression of the IL-1ra gene, depending on which IE genes are expressed in monocytes-macrophages. This study adds to the understanding of how HCMV can alter immune function during both active and latent infection.

The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

Cytomegalovirus (CMV) is an important cause of disease in the immunocompromised patient and CMV infection is associated with predominantly mononuclear inflammatory response. Since products of the CMV immediate early (IE) gene region are potent trans-activators, we used the monocyte cell line THP-1 and a transient transfection assay to determine if these viral proteins upregulate expression of the TNF gene. The IE genes of CMV upregulated TNF gene activity as judged by increases in promoter activity, steady state mRNA, and protein production. The presence or absence of the 3' untranslated region of the TNF gene did not affect gene expression induced by the IE gene products. These studies suggest that activation of TNF gene expression by the CMV IE gene products may, in part, account for the inflammatory response associated with CMV infections.

Cytomegalovirus immediate early genes prevent the inhibitory effect of cyclosporin A on interleukin 2 gene transcription.

The use of cyclosporin A (CsA) as an immunosuppressive agent has markedly improved the clinical outcome in solid organ transplantation. However, posttransplantation infection remains a significant problem and may contribute to subsequent organ rejection. In this study the effect of cytomegalovirus (CMV) immediate early (IE) gene products on interleukin 2 (IL-2) gene transcription in the absence and presence of CsA was investigated using a transient transfection system. Jurkat T cells were transfected with plasmids expressing the CMV IE gene products or with a control plasmid. The presence of the CMV IE2 gene product abolished the inhibitory effect of CsA on IL-2 promoter activation and gene transcription. This effect was noted regardless of the time of CsA addition relative to the time of stimulation and was independent of CsA concentration. CsA had no effect on the CMV or the IL-2 receptor promoters. These studies suggest that the CMV IE gene products may play a role in graft rejection after solid organ transplantation.

The immediate early genes of human cytomegalovirus upregulate expression of the cellular genes myc and fos.

Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (IE1 and IE2) can alter the regulation of the cellular immediate early genes (c-fos and c-myc). Plasmid constructs containing the promoter-regulatory regions c-myc or c-fos upstream of the reporter gene, chloramphemicol acetyl transferase, were co-transfected into T cells (Jurkat cells), monocytes/macrophages (THP-1 cells), or human fibroblast cells with plasmid constructs containing the promoter-regulatory region of the HCMV IE genes upstream of the bona fide IE1, IE2 or IE+2 genes; a plasmid that contained no IE coding region was used as a control. These studies show that both products of the HCMV IE genes markedly upregulated expression of the cellular c-fos and c-myc genes. The viral effects of individual proteins (IE1 or IE2) were dependent both on the promoter-regulatory region of the cellular gene and the cell type. In all cells, the combination of IE1 and IE2 further upregulated both cellular genes, suggesting a synergistic effect of IE1 with IE2. Both of the c-myc promoters (P1 and P2) were up-regulated by the HCMV IE gene products. IE1 and IE2 also upregulated the cells' endogenous c-myc and c-fos genes, as determined by amounts of the respective mRNAs. These studies show that HCMV can markedly alter cellular IE gene expression and that the effects of HCMV IE1 and IE2 proteins are dependent both on the promoter-regulatory region of the cellular gene and the type of cell in which the interaction occurs.

The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation.