IFN-gamma mRNA Research Articles

Immunological findings in 3 dogs clinically diagnosed with allergic rhinitis.

Three dogs clinically diagnosed with allergic rhinitis (AR) were examined for their immunological findings. House dust mites (HDM) such as Dermatophagoides farinae (DF) and D. pteronyssinus (DP) were identified as positive allergens in the 3 dogs with both intradermal skin test and serum antigen-specific IgE test. Lymphocyte blastogenic response of peripheral blood mononuclear cells (PBMCs) under stimulation with DF antigen in dogs with AR was higher than that in 4 healthy control dogs. Expression level of IL-4 mRNA in PBMCs obtained from the 3 AR dogs was higher than that in PBMCs obtained from 4 healthy control dogs before and after stimulation with DF antigen. Expression level of IFN-gamma mRNA in PBMCs was not different between the AR and control dogs before and after stimulation with DF antigen. These results suggested that allergic reaction to HDM antigen and T(H)2-type immune response were associated with the development of AR in 3 dogs examined in this study.

Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin.

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.

Augmented regeneration of partial liver allograft induced by nuclear factor-kappaB decoy oligodeoxynucleotides-modified dendritic cells.

To investigate the effect of NF-kappaB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft. Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GM-CSF-propagated DCs were treated with double-strand NF-kappaB decoy ODNs containing two NF-kappaB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A), GM-CSF -propagated DCs (group B), GM-CSF+IL-4 - propagated DCs (group C), and GM-CSF+NF-kappaB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h, respectively. Liver graft-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma level at the time of the maximal liver allograft regeneration were measured with (51)Cr release assay, semiquantitative RT-PCR and ELISA, respectively. Regeneration of liver allograft was markedly promoted by NF-kappaB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats. NF-kappaB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-gamma mRNA expression and serum IFN-gamma production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-gamma mRNA expression level and the serum IFN-gamma level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft. The data suggest that the augmented regeneration of partial liver allograft induced by NF-kappaB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liver-resident NK cells and the reduced IFN-gamma production.

Inhibitory Effect of Nicotine on Experimental Hypersensitivity PneumonitisIn VivoandIn Vitro

The incidence of hypersensitivity pneumonitis (HP) is lower in smokers than in nonsmokers. Because nicotine is immunosuppressive, we hypothesized that it could have a protective effect on HP induction in vivo. HP was induced in mice that were treated with nicotine either intraperitoneally (IP) (0.5 to 2.0 mg/kg/day) or intranasally (IN) (0.025 to 2.0 mg/kg/day). Both IP- and IN-treated animals had fewer bronchoalveolar lavage total cells and lymphocytes and a decreased lung tissue inflammation. IFN-gamma but not interleukin-10 mRNA expression was reduced in lung tissue of 2.0-mg/kg IN-treated animals. To test the effect of nicotine on alveolar macrophages, AMJ2-C11 cells were treated with nicotine and stimulated with lipopolysaccharide or Saccharopolyspora rectivirgula, a causative agent of HP. Nicotine reduced tumor necrosis factor release and tumor necrosis factor, interleukin-10, and IFN-gamma mRNA expression after stimulation and decreased CD80 expression by 55% in lipopolysaccharide-stimulated cells and by 41% in S. rectivirgula-stimulated cells. We conclude that nicotine could be, at least in part, responsible for the protection observed in smokers against HP. The inhibitory effect of nicotine on alveolar macrophages could be one of the mechanisms involved.

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.

Gamma interferon-mediated inflammation is associated with lack of protection from intravaginal simian immunodeficiency virus SIVmac239 challenge in simian-human immunodeficiency virus 89.6-immunized rhesus macaques.

Although gamma interferon (IFN-gamma) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-gamma-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-gamma T-cell responses and nonspecific IFN-gamma-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-gamma mRNA levels and a high frequency of IFN-gamma-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-gamma mRNA levels and strong in vitro SIV-specific IFN-gamma T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-gamma mRNA levels but weak in vitro anti-SIV IFN-gamma T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-gamma mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3(+) activated T cells. Thus, IFN-gamma-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-gamma-driven inflammation, but they did develop effective antiviral CD8(+)-T-cell responses.

High Expression of p40tax and Pro-inflammatory Cytokines and Chemokines in the Lungs of Human T-Lymphotropic Virus Type 1-Related Bronchopulmonary Disorders

Human T-lymphotropic virus type 1 (HTLV-1) is closely associated with the development of certain pulmonary diseases, such as bronchiolitis, although the pathologic mechanism remains unclear. To elucidate the pathogenesis of HTLV-1-associated bronchopulmonary disorders, we analyzed the relationship between expression of p40(tax), a regulatory component of HTLV-1 that stimulates various host genes, and synthesis of pro-inflammatory cytokines and chemokines by cells in BAL fluid (BALF) obtained from HTLV-1-infected patients. Reverse transcription-polymerase chain reaction was used to compare the expression of p40(tax) and pro-inflammatory cytokines and chemokines messenger RNA (mRNA) in BALF of 10 HTLV-1 carriers and 7 healthy subjects. We also studied the correlation between these parameters and the proportion of lymphocytes in BALF. The expression levels of pro-inflammatory cytokines (interferon [IFN]-gamma, interleukin-2) and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein [MIP]-1alpha, IFN-gamma-inducible protein-10 [IP-10]) were significantly higher in BALF of patients than of healthy subjects. The expression of IFN-gamma and MIP-1alpha mRNA correlated with that of p40(tax). IFN-gamma and IP-10 mRNA expression correlated with the proportion of lymphocytes in BALF. The percentage of lymphocytes in BALF increased with higher expression levels of p40(tax) mRNA, although the correlation was not significant. Our results suggested that p40(tax) seems be involved in the development of HTLV-1-associated bronchopulmonary disorders at least in part through the local production of pro-inflammatory cytokines and chemokines.

Neutrophils Sustain Pathogenic CD8 + T Cell Responses in the Heart

This study explores the influence of innate immunity on CD8(+) T-cell responses against heart tissue. Adoptive transfer of ovalbumin-specific CD8(+) effector T cells into CMy-mOva mice, which express ovalbumin in cardiac myocytes, results in a lethal acute myocarditis. The inflammatory infiltrate in the heart includes neutrophils as well as T cells. We used anti-Ly6G antibody to transiently deplete neutrophils at the time of onset of disease. By day 7 after receiving 5 x 10(5) CD8(+) effector T cells, 100% of control Ig-treated CMy-mOva mice had died, while 85% of anti-Ly6G-treated mice survived indefinitely. CD8(+) T-cell infiltration and tissue damage were present in both groups, but the disease was limited in the anti-Ly6G-treated mice, with a rapid disappearance of the adoptively transferred CD8(+) T cells within 11 days. Recovery occurred even though blood neutrophil counts began to rise 48 hours after the last anti-Ly6G treatment. Recovery was associated with a chronic CD4(+) cell infiltrate, and a rapid decline in expression of IFN-gamma and IP-10 mRNA in the myocardium. Neutrophil depletion did not effect survival of CMy-mOva mice that received 3 x 10(6) CD8(+) T cells. These data show that granulocytic inflammation sustains CD8(+) T-cell-mediated heart disease, which has important implications for the pathogenesis and treatment of acute myocarditis and allograft rejection.

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-γ production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.

Differential gene expression of cytokine and cell surface molecules in T cell subpopulation derived from mammary gland secretion of cows.

As T cell subpopulations in the mammary gland secretion (MGS) of cows dynamically vary through the lactation cycle, their functional analysis is important to understand the mammary immune responses. T cell subpopulations were positively selected from MGS during lactation period and non-lactation period (dry period) by a magnetic cell sorter. The messenger RNA (mRNA) expression of cytokine and cell surface molecules in the subpopulations stimulated with anti-CD3 was investigated using reverse transcription-polymerase chain reaction (RT-PCR). CD4+ T cells from MGS significantly expressed mRNA of interferon (IFN)-gamma, interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, IL-4, CD40 ligand (CD40L), Fas ligand (FasL) and IL-2 receptor (IL-2R) during dry period, and mRNA of IFN-gamma, IL-2 and TGF-beta during lactation period. Their expression during lactation period was always less than that during dry period. CD8+ T cells from MGS substantially expressed mRNA of IFN-gamma, IL-2, GM-CSF, TGF-beta, TNF-alpha, FasL and IL-2R during dry period and mRNA of IFN-gamma, GM-CSF, TGF-beta, TNF-alpha and c-kit during lactation period. The TGF-beta, TNF-alpha, c-kit and IL-2R mRNA expression of T cells in MGS during lactation period mostly depended on gammadelta T cells. Interestingly, c-kit mRNA was exclusively expressed in gammadelta T cells. The cytokine expression of T cells in MGS of cows depended on the T cell subpopulations. The present findings suggested that the activation of gammadelta T cells via c-kit receptor participated in the suppressed expression of cytokine mRNA in T cells during lactation period.

Mycobacterial infection in MyD88-deficient mice.

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.

Deficient IFN-gamma expression in umbilical cord blood (UCB) T cells can be rescued by IFN-gamma-mediated increase in NFATc2 expression.

Regulation of nuclear factor of activated T cells-c2 (NFATc2) gene expression is not clearly defined. We previously reported reduced NFATc2 protein expression in cord blood T lymphocytes. Here we show that NFATc2 expression in T cells is dependent in part on the presence of IFN-gamma during primary stimulation, as blocking of IFN-gamma blunted NFATc2 protein and mRNA upregulation. Conversely, addition of exogenous IFN-gamma during stimulation resulted in increased expression of NFATc2 in cord blood T lymphocytes. This correlated with rescue of deficient IFN-gamma expression by cord blood T cells. Rescue of IFN-gamma expression in cord blood T cells was dependent on the presence of antigen-presenting cells, as addition of IFN-gamma during stimulation of purified cord blood T cells did not result in an increase of IFN-gamma expression, and depletion of monocytes ablated the rescue of IFN-gamma expression. Our results point to impaired function in the antigen-presenting cell population of cord blood, playing a role in the hyporesponsiveness of T cells.

Further studies on the role of IL-12 in Pseudomonas aeruginosa corneal infection.

Previous studies have shown that in Pseudomonas aeruginosa ocular infection, IL-12 drives a Th1 T-cell response and IFN-gamma production in susceptible (cornea perforates) C57BL/6 (B6) mice, and that after similar infection of resistant (cornea heals) BALB/c mice, no IL-12 is detectable in cornea at either the mRNA or protein levels. Therefore, the purpose of this study was to test whether BALB/c mice are capable of responding to exogenous IL-12 administration, and whether disease responsiveness following P. aeruginosa challenge is modified. Immunostaining, RT/PCR, recombinant cytokine injection, and histopathology were used. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. Injection of BALB/c mice with recombinant (r) IL-12 converted these normally resistant animals to the susceptible phenotype as evidenced by corneal perforation within 5-7 days after infection. RT-PCR analysis of the corneas of rIL-12 vs PBS/BSA-treated mice showed a significant increase in IFN-gamma and TNF-alpha mRNA levels in the rIL-12 vs PBS/BSA (vehicle)-treated mice at 3 and 5 days p.i. In addition, similar analysis of IL-4 mRNA levels showed decreased amounts of the cytokine in rIL-12 vs vehicle-treated mice. Injection of rIL-4 into susceptible B6 mice, however, failed to rescue these animals from corneal perforation following P. aeruginosa challenge. These data provide evidence that BALB/c mice can respond to exogenous IL-12, that the cytokine promotes susceptibility by increasing IFN-gamma and TNF-alpha production, with a concomitant reduction in IL-4 levels; and that injected rIL-4 fails to rescue susceptible B6 mice from corneal perforation after bacterial challenge.

Marked Up-regulation of T Lymphocytes and Expression of Interleukin-9 in Bronchial Biopsies From Patients With Chronic Bronchitis With Obstruction

To examine the differences in the inflammatory cell and cytokine profile between patients with chronic bronchitis (CB) with and without airway obstruction compared to control subjects. We used bronchial biopsy samples from the patients and control subjects and analyzed them for the presence of CD3 T cells, CD68, major basic protein (MBP), elastase, and tryptase, as well as expression of messenger RNA (mRNA) coding for interleukin (IL)-4, IL-5, interferon (IFN)-gamma, IL-9, eotaxin, and IFN-gamma-inducible protein (IP)-10. The techniques of immunocytochemistry and in situ hybridization were used. Results were expressed as the number of immunoreactive and mRNA-positive cells per field. Increased number of elastase, CD68, and MBP-positive cells (n = 9, p < 0.01) was demonstrated in both groups of patients with CB compared to control subjects. In patients with CB and obstruction, the number of elastase, CD68, and the number of CD3-positive cells was significantly increased compared to patients with CB without obstruction (n = 9, p < 0.01). IFN-gamma mRNA expression was increased in both groups of patients with CB compared to control subjects (n = 9, p < 0.01). IL-9 mRNA was significantly increased only in patients with CB and obstruction (n = 9, p < 0.01). Co-localization studies demonstrated > 80% of all IL-9-positive cells to be CD3-positive T cells. IP-10 mRNA was significantly increased in both groups of patients with CB compared to control subjects (n = 9, p < 0.01). These results indicate a differential expression of inflammatory markers and cytokine mRNA in patients with obstructive CB. Increased presence of T lymphocytes and up-regulation of IL-9 and IP-10 mRNA expression in the bronchial biopsy samples may contribute to the airway obstruction in these patients.

Hyperinducible expression of the interferon-gamma (IFN-gamma) gene and its suppression in systemic lupus erythematosus (SLE).

Transient expression of IFN-gamma and IL-2 mRNA and its control by post-transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin-induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age-matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN-gamma gene expression in their PBMC to those showing moderate or low inducibility was increased five-fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL-2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN-gamma gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN-gamma mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (gamma-irradiation) or post-transcriptional down-regulation (cycloheximide). By contrast, patients with active SLE showing high IFN-gamma mRNA inducibility had normal suppressive capacity as well as post-transcriptional control. Hence, both high inducibility of the IFN-gamma gene and its suppression are relevant to disease. Hyperactivation of the IFN-gamma gene may be alleviated in mild SLE by a vigorous, concomitant activation of post-transcriptional control and of cell-mediated suppression.

Increased levels of IL-13 mRNA, but not IL-4 mRNA, are found in vivo in peripheral blood mononuclear cells (PBMC) of patients with atopic dermatitis (AD).

Previous studies using in vitro systems with various stimuli have shown that PBMC from patients with AD show increased levels of IL-4 but decreased levels of interferon-gamma (IFN-gamma) compared with PBMC from normal controls. However, in vitro conditions do not always mimic the in vivo condition. We therefore believe that it is important to quantify the expression of these cytokines in freshly isolated PBMC. This study examines the expression of IFN-gamma, IL-4 and IL- 13 mRNA in freshly isolated PBMC from adult patients with AD, from patients with psoriasis vulgaris and from healthy adults, using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Levels of IFN-gamma mRNA were significantly lower in PBMC of patients with AD than in controls. IL-4 mRNA levels did not differ significantly between groups. Conversely, levels of mRNA for IL-13 were significantly greater in PBMC of patients with AD than in controls. An increase in IL-13 expression may regulate the in vivo synthesis of IgE in patients with AD.

Cytokine responses to the native and recombinant forms of the major surface glycoprotein of Pneumocystis carinii.

Pneumocystis carinii is a major opportunistic pathogen and leading cause of morbidity in patients with AIDS. The major surface glycoprotein (MSG) of P. carinii, represented by a family of related proteins encoded by unique genes, is highly immunogenic and contains T cell-protective epitopes. We undertook the present study to define the CD4 T helper (Th) response by cytokine secretion to native MSG and a recombinant form of the protein, MSG-B. Spleen cells were collected from Lewis rats and restimulated with both native MSG and MSG-B. Within 24 h, the CD4 cells secreted high levels of interferon-gamma (IFN-gamma) in response to both types of antigen, indicative of a Th1 response; however, after 72h of incubation, only the native MSG stimulated secretion of IL-4 (Th2 response) from the cells. We then investigated whether the presence of IL-4 could alter the predominant Th1 phenotype by the CD4 cells in response to MSG and MSG-B. Cells cultured with native MSG and IL-4 produced low levels of IFN-gamma and elevated levels of IL-4. Interestingly, cells incubated with MSG-B and IL-4 reduced production of IFN-gamma, but were not stimulated to produce increased levels of IL-4. The presence of anti-IFN-gamma antibody in the MSG- or MSG-B-stimulated cultures did not effect the expression of IFN-gamma mRNA, suggesting that the generation of Th1 cells in response to MSG or MSG-B was not dependent on IFN-gamma. We conclude that native MSG, which contains multiple forms of this antigen, and recombinant MSG elicit different cytokine responses in vitro. These data are not only important to studies of MSG, but may also be relevant to the role of MSG in the immunopathogenesis of P. carinii infection in vivo.

The matrix protein pp65(341-350): a peptide that induces ex vivo stimulation and in vitro expansion of CMV-specific CD8+ T cells in subjects bearing either HLA-A*2402 or A*0101 allele.

The stimulation of PBMNCs with HLA Class I restricted synthetic peptides derived from CMV phosphorylated matrix protein 65 (pp65) evokes CMV-specific cytotoxic T lymphocyte (CTL) activity, a necessary condition for initiating adoptive immunotherapy against CMV-related diseases in immune-compromised patients. It was previously demonstrated that the CMV decamer (10-mer) peptide pp65(341-350), QYDPVAALFF, was able to induce CMV-specific CTLs in HLA-A*2402 CMV-seropositive individuals. We investigated the ability of the peptide pp65(341-350) to reactivate memory CD8+ T cells in CMV-seropositive subjects bearing either the HLA-A24 or A1 allele. CTL responses were measured by IFN-gamma mRNA expression and IFN-gamma protein production as well as cytotoxic activity. In this study it was found that peptide pp65(341-350) induced a specific reactivation of memory CD8+ T cells from CMV-seropositive donors expressing either HLA-A*2402 and/or HLA-A*0101. Moreover, a pp65(341-350)-specific selection and expansion using PBMNCs of CMV-seropositive donors bearing both HLA-A*2402 and HLA-A*0101 alleles produced cytotoxic CTLs to both HLA-A24 and A1 peptide-pulsed and autologous CMV-infected target cells. The results demonstrate that pp65(341-350) induced a specific CTL activity at both molecular and protein levels and that the peptide is specifically processed, presented, and recognized by subjects bearing HLA-A*2402 and/or A*0101. These findings suggest that it may be possible to use this single immune dominant peptide to induce and expand CMV-reactive CTLs for the treatment of individuals with both HLA-A24 and A1 types.

Disruption of TGF-beta signaling in T cells accelerates atherosclerosis.

Increasing evidence suggests that atherosclerosis is an inflammatory disease promoted by hypercholesterolemia. The role of adaptive immunity has been controversial, however. We hypothesized that proatherogenic T cells are controlled by immunoregulatory cytokines. Among them, TGF-beta has been implied in atherosclerosis, but its mechanism of action remains unclear. We crossed atherosclerosis-prone ApoE-knockout mice with transgenic mice carrying a dominant negative TGF-beta receptor II in T cells. The ApoE-knockout mice with disrupted TGF-beta signaling in T cells exhibited a sixfold increase in aortic lesion surface area, a threefold increase in aortic root lesion size, and a 125-fold increase in aortic IFN-gamma mRNA when compared with age-matched ApoE-knockout littermates. When comparing size-matched lesions, those of mice with T cell-specific blockade of TGF-beta signaling displayed increased T cells, activated macrophages, and reduced collagen, consistent with a more vulnerable phenotype. Ab's to oxidized LDL, circulating T cell cytokines, and spleen T cell activity were all increased in ApoE-knockout mice with dominant negative TGF-beta receptors in T cells. Taken together, these results show that abrogation of TGF-beta signaling in T cells increases atherosclerosis and suggest that TGF-beta reduces atherosclerosis by dampening T cell activation. Inhibition of T cell activation may therefore represent a strategy for antiatherosclerotic therapy.

IFN-γ–RegulatedToxoplasma gondiiDistribution and Load in the Murine Eye

To establish a mouse model of ocular toxoplasmosis in both wild type (WT) and immunocompromised hosts and to clarify the effects of interferon (IFN)-gamma on the infectivity of Toxoplasma gondii in various parts of the eye. Susceptible WT C57BL/6, resistant WT BALB/c, and IFN-gamma knockout (GKO) mice were infected with cysts of T. gondii perorally. The tissues were harvested for molecular and histopathologic studies. Analysis included a quantitative competitive polymerase chain reaction (QC-PCR) assay and reverse transcription (RT)-PCR for IFN-gamma and stage conversion markers. All animals underwent ophthalmic examinations including fluorescein angiography (FA). In WT C57BL/6 mice, T. gondii was detected in tissue in the following order: brain, retina, choroid, sclera, and optic nerve (ON). The highest T. gondii load was observed in the posterior retina, and was much greater than that in WT BALB/c mice. In GKO mice, disseminated infection was evident, and the T. gondii load was highest in the choroid and ON. IFN-gamma mRNA expression in WT C57BL/6 mice was higher than that in WT BALB/c mice after infection. Tachyzoites existed in GKO mice, whereas bradyzoites existed in WT C57BL/6 mice. FA showed dye leakage from the retinal capillaries of GKO mice. The T. gondii load in the retina in the susceptible WT strain continued to increase, unlike in the resistant WT strain. IFN-gamma was shown to regulate the T. gondii load and interconversion in the eye. A toxoplasmic vasculitis model was established with GKO mice and assay systems with QC-PCR and FA.