Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.

Prokaryotic Expression, In Vitro Biological Analysis, and In Silico Structural Evaluation of Guinea Pig IL-4.

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

Expression of interferon-gamma and tumour necrosis factor-alpha messenger RNA does not correlate with protection in guinea pigs challenged with virulent Mycobacterium tuberculosis by the respiratory route.

Cytokine messenger RNA (mRNA) expression was investigated in the spleen and lung digest cells of bacillus Calmette-Guérin (BCG)-vaccinated and non-vaccinated guinea pigs following low-dose, pulmonary exposure to virulent Mycobacterium tuberculosis. After purified protein derivative (PPD) stimulation, the levels of lung cell interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and spleen cell interleukin-12 (IL-12) p40 mRNAs were significantly increased in the non-vaccinated M. tuberculosis-infected guinea pigs compared to the BCG-vaccinated guinea pigs. In contrast, the expression of anti-inflammatory transforming growth factor-beta and IL-10 mRNAs was significantly enhanced in the spleens of BCG-vaccinated animals. Despite the presence of protective cytokine mRNA expression, the non-vaccinated guinea pigs had significantly higher lung and spleen bacterial burdens. In contrast, BCG-vaccinated guinea pigs controlled the bacterial multiplication in their lungs and spleens, indicating that both protective as well as anti-inflammatory cytokine responses are associated with a reduction in bacteria. In addition, lung digest cells from non-vaccinated guinea pigs contained a significantly higher percentage of neutrophils, CD3(+) and CD8(+) T cells, while the percentage of macrophages was increased in the BCG-vaccinated animals. Total and purified lung digest T cells co-cultured with lung macrophages (LMøs) proliferated poorly after PPD stimulation in both non-vaccinated and BCG-vaccinated animals while robust proliferation to PPD was observed when T cells were co-cultured with peritoneal macrophages (PMøs). Macrophages within the lung compartment appear to regulate the response of T cells irrespective of the vaccination status in guinea pigs. Taken together, our results suggest that type I cytokine mRNA expression is not associated with vaccine-induced protection in the low-dose guinea pig model of tuberculosis.

Differential activation of alveolar and peritoneal macrophages from BCG-vaccinated guinea pigs.

We compared the effect of BCG vaccination on the mRNA expression of two prototypic cytokines, IL-12 (Type 1) and IL-10 (Type 2), in guinea pig resident alveolar macrophages (AM) or resident peritoneal macrophages (PM). Cells were stimulated with live or heat-killed Mycobacterium tuberculosis, and/or with recombinant guinea pig (rgp) TNF-alpha and/or rgp IFN-gamma. AM from BCG-vaccinated guinea pigs expressed significantly less IL-10 mRNA and more IL-12p40 mRNA compared to AM from naive animals following stimulation with heat-killed mycobacteria. In PM from BCG-vaccinated guinea pigs, IL-12p40 mRNA was significantly up-regulated; however, the level of IL-10 mRNA was not affected by prior vaccination. rgp TNF-alpha or rgp IFN-gamma, both alone and together, induced a significant increase of H(2)O(2) production in PM from BCG-vaccinated animals. MHC class II expression was dramatically up-regulated in PM from BCG-vaccinated animals stimulated with both rgp TNF-alpha and rgp IFN-gamma. The levels of IL-10 and IL-12p40 mRNA were significantly enhanced in PM stimulated with combinations of rgp TNF-alpha and rgp IFN-gamma, and those cells suppressed the intracellular accumulation of viable, virulent M. tuberculosis. BCG vaccination results in the differential activation of guinea pig AM and PM to promote a Type 1 cytokine milieu and control intracellular mycobacteria.

Cloning and characterization of guinea pig CXCR1.

IL-8/CXCL8 plays a critical role in the trafficking and activation of neutrophils via its receptors, CXCR1 and CXCR2, in humans. CXCR1 is highly selective for IL-8, whereas CXCR2 is activated by all CXC chemokines with an ELR motif. In mice and rats, neither IL-8 nor CXCR1 is present, making it difficult to evaluate the in vivo roles of the IL-8/CXCR1 interactions. We previously demonstrated the presence of IL-8 in the guinea pig (gp), suggesting that its specific receptor CXCR1 is also present in this species. Here, we obtained two gp genomic DNA clones, clones 8 and 10, coding for the potential orthologues of CXCR1 and CXCR2, respectively. Transcripts for these genes were expressed in neutrophils, but not in macrophages. Functionally, both gp and human (h) IL-8 induced cell migration and ERK phosphorylation in HEK 293 cells expressing either receptor, whereas hGRO activated only cells expressing the clone 10 protein, confirming that clone 8 indeed coded for gpCXCR1. 125I-labeled hIL-8 bound to gpCXCR1 and addition of unlabeled hIL-8 completely abolished the binding; however, unlabeled gpIL-8 failed to compete against 125I-labeled hIL-8, strongly suggesting that the avidity of hIL-8 to gpCXCR1 is higher than that of gpIL-8. Identification and characterization of CXCR1 in the guinea pig will allow us to use this small animal model to evaluate the role of the IL-8/CXCR1 interactions and to examine the efficacy of CXCR1 antagonists in vivo.

Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs.

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.

A new assay system for guinea pig interferon biological activity.

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Prokaryotic expression and in vitro functional analysis of IL-1ß and MCP-1 from guinea pig.

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1ß was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

Guinea pig skin, a model for epidermal cellular and molecular changes induced by UVR in vivo and in vitro: effects on Mycobacterium bovis Bacillus Calmette-Guérin vaccination.

Previously, we reported that ultraviolet B-radiation (UVR) suppressed Bacillus Calmette-Guérin (BCG) vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs (GP). Herein, we investigated the cellular and molecular changes within the irradiated GP epidermis and the in vivo effect of supernatants from UV-irradiated (200 J m(-2)) epidermal cells (UV-sup) on M. bovis BCG vaccination. UVR increased the number of nucleated keratinocytes in the skin, but caused a decrease in the proportions of CD25(+)T cells. In the spleen, UVR resulted in a decrease in the proportions of T-cell subsets including CD25(+)T cells, and major histocompatibility complex (MHC) class II(+) and CD14(+) cells. Similarly, significant up-regulation of several cytokine mRNAs including IL-10 was also observed. Furthermore, UV-sup significantly reduced the MHC class II expression in peritoneal cells and reduced T-cell proliferation to ConA. The proliferation to purified protein derivative (PPD) was restored to normal levels by anti-IL-10 antibody. The UV-sup when injected into BCG-vaccinated GP significantly diminished the skin test response and T-cell proliferation to PPD and up-regulated the expression of IL-10, IL-4, IL-1ß and Foxp3 mRNAs in the lymph node or spleen. Thus, whole body UVR induces profound cellular and molecular changes and injection of UV-sup from epidermal cells mimics the effect of whole body UVR in BCG-vaccinated GP.

Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17.

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

Molecular cloning and expression of the IL-10 gene from guinea pigs.

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.

Cloning of guinea pig IL-4: reduced IL-4 mRNA after vaccination or Mycobacterium tuberculosis infection.

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model.

BCG vaccination in the cotton rat (Sigmodon hispidus) infected by the pulmonary route with virulent Mycobacterium tuberculosis.

To evaluate the usefulness of the American cotton rat (Sigmodon hispidus) in the evaluation of vaccine-induced resistance, we infected BCG-vaccinated and non-vaccinated cotton rats with Mycobacterium tuberculosis (H37Rv) via the respiratory route. Lung histopathology of these animals showed loose, disorganized granulomas which were non-necrotic up to 8 weeks post-infection. Moreover, we were not able to detect a DTH response after intradermal injection with PPD antigen. Prior BCG vaccination significantly reduced lung and spleen bacterial loads by 1-1.5log CFU and upregulated PPD-induced proliferation and production of IFNgamma in lymphocyte cultures. We conclude that pulmonary infection of the cotton rat with Mtb more closely resembles the phenotype seen in mice rather than guinea pigs.

Neutralization of TNFalpha alters inflammation in guinea pig tuberculous pleuritis.

Previously, treatment with anti-gpTNFalpha antibody enhanced TNFalpha mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFalpha or normal serum control. Neutralizing TNFalpha reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell-associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFalpha antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFalpha and IFNgamma mRNA. By day 5, treatment with anti-gpTNFalpha antibody significantly reduced TNFalpha mRNA and increased TGFbeta and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFalpha mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFalpha, IFNgamma, and TGFbeta mRNA dominated the anti-gpTNFalpha-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFbeta mRNA. Neutralization of TNFalpha hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.

Ultraviolet radiation reduces resistance to Mycobacterium tuberculosis infection in BCG-vaccinated guinea pigs.

Ultraviolet radiation (UVR) in sunlight suppresses anti-microbial immunity and may impair vaccine inefficacy. To investigate whether UVR reduces the protective efficacy of BCG vaccine, guinea pigs were exposed to a single dose of 5.6, 11.2, or 22.5 kJ/m(2) of UVR either 3 days before or 3 or 28 days after BCG vaccination and then challenged by the aerosol route with virulent Mycobacterium tuberculosis 6 weeks later. Pre-challenge skin test responses to purified protein derivative (PPD) were significantly reduced in the UV-irradiated animals. Similarly, exposure to UVR reduced the proliferation of spleen cells to PPD, altered cytokine mRNA expression in spleen and lung digest cells, and increased the colony forming units (CFU) in the lungs of M. tuberculosis-infected guinea pigs at 5 weeks post-infection. Spleen IFN-gamma, TNF-alpha, IL-10 and IL-12p40 mRNA levels were higher when exposed to 5.6 kJ/m(2) UVR, while the levels were significantly reduced at 11.2 and 22.5 kJ/m(2). Lung cell IL-10 mRNA expression increased at all UVR doses. Thus, UVR exposure a few days before or after BCG vaccination at a distant unirradiated site impaired vaccine-induced resistance against virulent M. tuberculosis in guinea pigs possibly by altering cytokine responses in a UVR dose-dependent manner.

Altered inflammatory responses following transforming growth factor-beta neutralization in experimental guinea pig tuberculous pleurisy.

The predominant extrapulmonary form of tuberculosis, which develops in 10% of diseased individuals, is pleurisy. The immune response mounted against Mycobacterium tuberculosis in the pleural cavity is one that is sufficient for clearing the organism without therapeutic intervention. Thus, examining the role of immune constituents in this context will provide understanding of the vital role they play in controlling tuberculosis. In this study, experimental tuberculous pleurisy was induced in guinea pigs, and anti-TGF-beta was administered intrapleurally to the guinea pigs daily throughout the study (8 days). Neutralizing TGF-beta resulted in a significant reduction in the percentage of lymphocytes and CD8+ cells present in the pleural exudate, decreased proliferative responses of pleural cells to ConA and PPD, and decreased mRNA expression of IFN-gamma and CCL5 in pleural effusion cells. Conversely, the percentage of neutrophils was significantly increased in anti-TGF-beta-treated guinea pigs, along with upregulated mRNA expression of CXCL8. The percentage of macrophages in the pleural exudate, TNF-alpha and IL-12p40 mRNA expression, and the histopathological response were not significantly altered. While TGF-beta is generally thought of as an immunosuppressive cytokine, the results of this study demonstrate its importance in promoting an inflammatory response, and highlight its bipolar nature.

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria.

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.

Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination.

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone. Compared to the IFN-gamma mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.