Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells.

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.

Human immunodeficiency virus type 1 and mucosal humoral defense.

Mucosal sites serve as the principle venues through which primary human immunodeficiency virus type 1 (HIV-1) infections are transferred from donor to host. These moist tissues, which provide the interface with the external environment, also provide access to many of the secondary opportunistic infections that aggravate and may accelerate HIV-1 disease. Antibodies to HIV-1, particularly of the IgG rather than the IgA class, have been detected in virtually all mucosal fluids from HIV-1-infected patients. However, the ability of such patients to generate de novo humoral responses to new mucosal pathogens is impaired. Current studies are directed to characterizing the functional role of natural and infection-derived antibodies in control of HIV-1 infection as well as the impact of HIV-1 disease on mucosal B cell responses to immunization and infection.

Coincidental changes in behavior and plasma cortisol in unrestrained pigs after intracerebroventricular injection of tumor necrosis factor-alpha.

The coincidental behavioral and physiological responses to inflammatory stimuli administered either peripherally or centrally were evaluated. In the first study, twenty castrated male pigs were injected ip with 0, 0.5, 5, or 50 microg/kg BW lipopolysaccharide (LPS). Body temperature was monitored telemetrically, and serial blood samples were collected via an indwelling jugular catheter for determination of plasma cortisol and tumor necrosis factor-alpha (TNF-alpha) concentrations. Sickness behaviors were measured during 10-min tests at 0, 2, 4, 8, 12, and 24 h post injection. The 5 and 50 microg/kg doses of LPS increased plasma concentrations of cortisol and TNF-alpha, while inducing anorexia, hypersomnia, and fever. In contrast, although 0.5 microg/kg LPS induced acute anorexia, hypersomnia, and fever, it did not increase plasma TNF-alpha; and the cortisol response was small and transient, suggesting the behavioral system in pigs is more responsive to LPS than the hypothalamic-pituitary-adrenal (HPA) axis. Because LPS-induced behavior and activation of the HPA axis involve proinflammatory cytokines in the brain, in a second study, unrestrained pigs with jugular catheters were injected intracerebroventricularly (I.C.V.) with recombinant porcine TNF-alpha. Vehicle or TNF-alpha (0, 5, or 50 ng/kg) was injected I.C.V., and plasma cortisol and behavior were determined as before. Pigs injected I.C.V. with 50 ng/kg TNF-alpha showed anorexia, hypersomnia, and an abrupt increase in plasma cortisol concentration. Whereas 5 ng/kg TNF-alpha I.C.V. also induced marked sickness behavior, it failed to stimulate the HPA axis, as indicated by plasma cortisol levels. That there was a distinct difference in the magnitude of behavioral and endocrine responses to LPS and TNF-alpha suggests that different systems that are responsive to inflammatory stimuli exhibit different sensitivities.

Inflammatory cytokines in animal health and disease.

Inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and IL-8, are rapidly induced early in a disease or injury process. They mediate and modulate myriad healing processes but, if overexpressed, may exacerbate the severity of a disease condition. In order to test this concept and to establish a foundation for the role of inflammatory cytokines in the pathogenesis of gram-negative bacterial infections in the respiratory tract of animals, the patterns of inflammatory cytokine expression were determined in experimental porcine pleuropneumonia. We observed that IL-1 and IL-6, but not TNF, were rapidly and dramatically elevated in the lavage fluid of the lung within 24 h of infection. The increased levels of IL-1 might contribute to increased severity of disease, but elevated IL-6 levels were consistent with a protective acute phase response. Additional studies were performed to examine the hypothesis that IL-4 expression later in infection might be involved in turning off the inflammatory response and promoting an antigen-specific humoral immune response. Interleukin-4 efficiently suppressed inflammatory cytokine production in alveolar macrophages. Its expression was induced in peripheral blood mononuclear cells by TNF, IL-4, and by reexposure to a specific antigen. To obtain the maximum amount of information on the role of inflammatory cytokines in animals of veterinary significance it will be useful to perform studies in species such that evolutionary relatedness will allow widespread application of the findings. Furthermore, the variety of molecules involved in inflammatory cytokine regulation will require much more extensive investigations of the relevant enzymes, inhibitors and receptors in veterinary species. Finally, the complexity and redundancy of immune defenses in animals mean that attempts to modulate health status through manipulation of inflammatory cytokines must be performed with caution and that a multiplicity of processes will be affected.

Regulation of interleukin-6 expression in porcine immune cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, acute-phase reaction, and hematopoiesis. As a first step in studying the actions of IL-6 in pigs, the regulation of IL-6 expression was examined in various swine cells, including a fibroblast cell line, peripheral blood mononuclear cells (PBMC), and alveolar macrophages. IL-6 expression in transformed swine testicular (TST) fibroblasts was enhanced by TNF and IL-1 beta and to a lesser extent by poly(I).(C) and LPS. IL-6 was induced in porcine PBMC by either LPS or PHA; however, the combination of LPS plus PHA resulted in maximal IL-6 expression. Furthermore, in PBMC cells separated by adherence, LPS was a more potent inducer than PHA in adherent cells, whereas PHA was more potent in nonadherent cells. Alveolar macrophages collected from different pigs could be divided into low and high responders with respect to IL-6 induction by LPS. IL-6 mRNA induction by LPS could be detected in only 6 of 20 donor animals. Other inflammatory cytokines (IL-8, IL-beta, and TNF) were readily induced by LPS in alveolar macrophages from both low and high responders. Treatment of low-responder alveolar macrophages with conditioned medium containing IFN-gamma did not significantly alter the capacity of these macrophages to synthesize IL-6 mRNA in response to LPS. Comparison of IL-6 production capacity by the cell types in this study revealed the following order: PBMC = high-responder alveolar macrophages >> TST.cells > low-responder alveolar macrophages. Thus, PBMC appear to be quantitatively the most significant source of IL-6 in swine on a per cell basis.

Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae.

An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.

Interleukin-4 suppresses inflammatory cytokine gene transcription in porcine macrophages.

Inflammatory cytokines, including interleukin (IL)-1 alpha, IL-1 beta, IL-8, and tumor necrosis factor alpha (TNF-alpha) are produced by macrophages in response to a variety of pathogenic stimuli. We show here that the expression of inflammatory cytokines is suppressed by IL-4 at the transcriptional level. Interleukin-4, when added together with bacterial lipopolysaccharide (LPS), suppressed LPS-induced increases in mRNA levels of IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha in alveolar macrophages. The level of suppression was dependent on dose and time of exposure and reached a maximum of 75-80% of uninduced values for IL-1 alpha, IL-8, and TNF. Interleukin-1 beta expression was completely inhibited by IL-4. The amount of secreted protein, as determined by TNF-alpha bioassay, was also suppressed by IL-4. Half-maximal suppression occurred at IL-4 concentrations between 0.02 and 0.1 ng/ml for all inflammatory cytokines. Nuclear run-on assays showed that IL-4 suppressed transcriptional activity of all inflammatory cytokines. Messenger RNA stability was not changed by IL-4. The data suggest that IL-4 plays an important transcriptional role in the regulation of alveolar macrophage inflammatory activities in respiratory disease and raise the possibility that IL-4 may function in vivo as a coordinator of inflammatory and immune responses.

Regulation of interleukin-8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide.

Interleukin (IL)-8 is a macrophage-derived neutrophil chemotactic factor that plays an important role in the recruitment of neutrophils to inflammatory loci. Hence, expression of IL-8 by alveolar macrophages may be a significant factor in host defense in the lung and in the pathogenesis of pneumonia in swine. To initiate molecular studies of IL-8 regulation in pigs, we cloned IL-8 cDNA and examined the regulation of its mRNA in alveolar macrophages. The porcine IL-8 cDNA consists of 1491 base pairs including a coding region of 309 base pairs. The deduced amino acid sequence was 75 and 81% similar to human and rabbit IL-8, respectively. Resting macrophages contained low levels of IL-8 mRNA, which increased markedly after exposure to bacterial lipopolysaccharide (LPS). LPS induction of IL-8 was direct, not mediated through elevation of tumor necrosis factor or interleukin-1. The effect of LPS on IL-8 expression was dose dependent, and induction was observed at a concentration of 10 pg/ml. IL-8 mRNA expression was detectable within 0.5 h after stimulation with LPS, peaked at 3-6 h at about 30-fold higher levels than in resting cells, and was maintained for 24 h. Secreted IL-8, measured by neutrophil chemotaxis, was induced within 4 h by LPS, and accumulated in the media throughout the 24-h period. The mechanism of induction of IL-8 mRNA appeared to involve transcription and RNA processing. Nuclear run-on analysis showed that the IL-8 gene was actively transcribed in noninduced cells; upon stimulation with LPS, the rate of IL-8 transcription was increased about 4-fold. A single mature mRNA species was detected by primer extension analysis. The half-life of IL-8 mRNA transcripts in aveolar macrophages was approximately 2 h and did not change after LPS stimulation. The ability of LPS to induce IL-8 expression was suppressed by recombinant human IL-4 and dexamethasone in a concentration-dependent manner. These observations indicate that the expression of IL-8 is an early event in the sequelae to bacterial infection in the lung.

Characterization of transforming growth factor-beta 1 gene expression in porcine immune cells.

We have investigated the regulation of transforming growth factor beta 1 gene expression in a variety of porcine immune cell populations, including peripheral blood mononuclear cells (PBMC), peripheral blood monocytes, alveolar macrophages and lymphoid cells from various swine lymphoid tissues. Using porcine transforming growth factor beta 1 cDNA probes in Northern blot assays, messages of 2.5 and 3.5 kb TGF beta 1 mRNA were detected in the cells investigated. A variety of mitogenic and immunomodulatory substances were examined for their ability to induce TGF beta 1 mRNA expression. These include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS), dexamethasone (Dex), tumor necrosis factor (TNF) and interleukin (IL)-1 alpha. While low level constitutive expression of TGF beta 1 mRNA was detected from all cells investigated, PMA treatment of PBMC and alveolar macrophages resulted in a more than 10-fold increase in the steady-state level of TGF beta 1 mRNA within 2 hr of PMA addition. Also, the effect of opiate drugs, methadone (Md) and morphine (Mor), on TGF beta 1 gene expression was determined. Cells treated with opiates expressed the same levels of TGF beta 1 mRNA as untreated cells. Since TGF beta 1 biological activity can be induced by opiates, the regulation of TGF beta 1 gene expression likely involves mechanisms that do not cause changes in mRNA levels.