IFN-γ Positive Cells Research Articles

Exacerbation of DSS induced colitis by localized delivery of IFN-β secreted by Lactobacillus acidpohilus (47.6)

Abstract There have been conflicting reports of the role of Type I interferons in gut inflammation and inflammatory bowel diseases. While in some patients with active ulcerative colitis (UC), treatment with Type I interferons induced remission of symptoms, other reports have documented the spontaneous occurrence of UC in multiple sclerosis and chronic hepatitis C patients receiving IFN-β therapy. In order to evaluate the potential for IFN-β as a therapeutic for IBD, we developed a delivery system involving probiotic bacteria. Treatment of healthy mice with Lactobacilli secreting IFN-β results in increased IFN-γ positive cells within the gut as well as a reduction in the percentage of T-regulatory cells. When mice were pretreated with Lactobacilli secreting IFN-β prior to induction of acute DSS colitis, we observed more weight loss and a worsening of intestinal thickening/shortening. In addition, there was an increase in the percentage of Th17 cells, as well as an increase in the production of TNF-α by colonic tissue. This study demonstrates that localized delivery of IFN-β by probiotic bacteria results in an exacerbation of DSS induced colitis in a preventative model.

Constitutive expression of interferons in swine leukocytes

Interferon (IFN)-α and IFN-γ positive cells were revealed by flow cytometry in peripheral blood mononuclear cells (PBMC) of Specific Pathogen Free (SPF) pigs. A low prevalence of IFN-γ positive cells was also detected in PBMC of some Porcine Reproductive and Respiratory Syndrome virus-infected pigs and uninfected, control pigs. IFN-α positive cells showed phenotypes of both monocytes and plasmacytoid dendritic cells. The presence of IFN-α in PBMC was also confirmed by Western blotting. By immunoprecipitation, IFN-α was detected as 32 and 55–57 kDa bands in PBMC of healthy SPF piglets. These samples were also IFN-γ positive; the cytokine was revealed as 24, 37 and 54 kDa bands. The unusual molecular mass values of intracellular interferons were probably due to oligomerization, as previously described for human IFN-α. Swine intracellular IFN-α displayed the expected antiviral activity on bovine MDBK cells. The results indicate that interferons are constitutively expressed in swine leukocytes with peculiar molecular features.

An intrinsic role for the alternative NF-kappa B pathway in the T-cell response to virus (35.21)

Abstract Regulation of NF-κB mediated signaling is controlled by two systems: the classical pathway and the alternative pathway. The alternative pathway refers to the processing of p100 to p52 regulated by an enzyme called NF-κB inducing kinase (NIK), and is essential for the normal function of B-cells, macrophages, and dendritic cells; however, little is known about its function in T-cells. Preliminary experiments determined that activation of the alternative pathway is necessary for CD4 mediated graft versus host disease. Based on those findings, we hypothesized that the broader T-cell response to a viral infection is also dependent on the alternative NF-κB pathway. Because of the dominant effects of deficiencies in the alternative pathway on B-cells, macrophages and dendritic cells, we could not discretely assess the T-cell response to pathogen in alternative pathway mutants. Therefore, we generated mixed bone marrow (BM) chimeras using BM from wild-type mice and alymphoplasia (aly) mice that lack a functional NIK protein or BM from p100 knockout mice. In these chimeras, 15-20% of the T-cells, less than 2% of the B-cells and less than 2% surveyed myeloid cells, possessed the mutant genotype. This allowed us to assess the function of T-cells lacking the alternative pathway in an otherwise normal immune system. Chimeras were infected with lymphocytic choriomeningitis virus (LCMV) and the immune response was analyzed 8 days post-infection. The percent of responding CD8 and CD4 T-cells was significantly reduced among aly/aly and p100-/- populations compared to wild-type cells, as measured by the percent of IFNγ positive cells following ex vivo stimulation with LCMV peptides and activation marker expression. These data demonstrate a T-cell intrinsic role for the alternative NF-κB pathway in the immune response to a viral infection.

Study of some of the mechanisms involved in the prevention against Salmonella enteritidis serovar Typhimurium infection by lactic acid bacteria

The possible mechanism exerted by different lactic acid bacteria (LAB) in the protection against Salmonella enteritidis serovar Typhimurium (S. typhimurium) infection was determined. LAB was administered to BALB/c mice, and the animals were subsequently challenged with S. typhimurium. The inhibition of the translocation of S. typhimurium in the liver was correlated with a decrease in cellular apoptosis determined in slices from the small intestine of mice. The microbiocidal activity of peritoneal macrophages was increased by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but not for the probiotic strain L. casei CRL 431. The levels of IFNγ and Bcl-2 positive cells in the small intestine of mice fed with the LAB were also determined by immunofluorescence. Using in vivo studies, we demonstrated that the biological and immune mechanisms induced by the LAB studied were different for each bacterium and were mediated by anti-S. typhimurium S-IgA microbiocidal activity and/or cellular apoptosis inhibition of infected immune cells.

IL-18 deficiency inhibits both Th1 and Th2 cytokine production but not the clinical symptoms in experimental autoimmune neuritis

IL-18 deficient (IL-18 −/−) mice were used to investigate the role of IL-18 in the pathogenesis of experimental autoimmune neuritis (EAN) which was induced by immunization of the mice with P0 protein peptide 180–199. The clinical course was not different between IL-18 −/− and wild-type mice. The splenic mononuclear cell (MNC) proliferation was also similar in both animal groups. However, the percentages of IFN-γ, IL-10 and IL-12 positive cells were decreased among infiltrating MNC of cauda equine in IL-18 −/− mice. This indicates that IL-18 deficiency inhibits the production of both Th1 and Th2 cytokines in the target organ of mice with EAN.

Roles of osteoblasts, osteoclasts, T cells and cytokines in glucocorticoid-induced osteoporosis

Steroid-induced osteoporosis is the most common form of secondary osteoporosis. The mechanisms of glucocorticoid (GC)-induced osteoporosis may be divided into indirect and direct effects. A putative indirect mechanism of GC-induced osteoporosis is thought to be secondary hyperparathyroidism or estrogen deficiency. Recent studies, however, suggest that the direct action of GC on bone metabolism is more important. GC stimulates the expression of the receptor activator of NF-κB ligand (RANKL) and inhibits osteoprotegerin (OPG) expression in a dose-dependent manner in human osteoblasts. The resultant increase in the RANKL:OPG ratio results in the induction of osteoclastogenesis. GC predominantly inhibits human osteoblast proliferation and enhances the differentiation of human-pre-osteoblasts. GC also acts directly on human osteoblasts to up-regulate the expression of M-CSF which is an essential cytokine for the survival of osteoclast precursors. The addition of GC to the culture of human peripheral blood mononuclear cells (PBMC) results in a marked increase in the formation of osteoclasts and an increase in lacunar resorption. Moreover, GC has been shown to inhibit apoptosis in mouse osteoclasts. In addition, the direct effect of dexamethasone (Dex) upon human osteoclastogenesis from PBMC is mediated via the balance between RANKL and IFN-γ in activated CD4 T cells. Dex induced human osteoclastogenesis without adding osteoblasts or soluble RANKL. Dex dose-dependently increased the ratio of RANKL-positive cells to IFN-γ positive cells (RANKL:IFN-γ ratio) only by reducing IFN-γ-positive cells, suggesting that Dex stimulates osteoclast differentiation by elevating the RANKL:IFN-γ ratio in CD4+ T cells. The balance between RANKL and OPG (RANKL:OPG) or RANKL and IFN-γ (RANKL:IFN-γ) on osteoblasts and T cells may have an important role in GC-induced osteoclastogenesis. Thus, therapeutic modulation of the expression of RANKL, OPG and IFN-γ by osteoblasts and T cells represents a novel strategy to prevent GC-induced osteoporosis.

Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4 + T cells in humans

Several alterations in immune function and a concomitant progressive increase in pro-inflammatory status are the major characteristics of ageing process. Cytokines play a key role during ageing acting both in regulatory communication among cells and in effector activity during an immune response. The impact of age on intracellular Type 1 (IFN-γ and TNF-α) and Type 2 (IL-4) cytokines, after stimulation with PMA/ionomycin, was determined in three CD4 + T subsets, i.e. CD95 −CD28 + (virgin), CD95 +CD28 + (activated/memory), and CD95 +CD28 − (effector/memory) from 47 subjects aged between 21 and 99 years. The percentage of IFN-γ positive cells significantly decreased in virgin CD4 + subset both in old and nonagenarian subjects, as well as in activated/memory T cells from old in comparison with young subjects. The percentage of TNF-α positive cells significantly decreased in activated/memory CD4 + subset from old people. Regarding Type 2 cytokines, IL-4 positive cells significantly increased in activated/memory CD4 + subset from nonagenarians. On the whole our data indicate that: (1) different Type 1 and Type 2 cytokine-positive CD4 + T subsets are differently affected by ageing process; (2) activated/memory T cells appear to be the most affected subset; (3) a shift towards an increased role of Type 2 cytokines and a diminished role of Type 1 cytokines emerges with ageing.

Th1/Th2 cytokine pattern in bronchoalveolar lavage fluid and induced sputum in pulmonary sarcoidosis

BackgroundSarcoidosis is thought to be a T-helper type 1 cytokine (Th2 cytokine) mediated disorder. Induced sputum (IS) has been proposed as a useful non-invasive method, mainly for the assessment of the airway diseases. The aim of this study was to explore induced sputum (IS) CD4+Th1 T-lymphocyte subpopulation and to compare them with those of bronchoalveolar lavage fluid (BALF) in patients with sarcoidosis.MethodsWe studied prospectively 21 patients (12 female, 9 male) of median age 46 yr (range, 25–65) with sarcoidosis and 10 normal subjects (5 female, 5 male) of median age 39 yr (range, 26–60). IS was performed with hypertonic saline solution using an ultrasonic nebulizer. BALF was performed within 10 days of IS. After stimulation of sputum lymphocytes with phorbol-myristate-acetate, we used double immunocytochemical methods to identify CD4+ IFN-γ positive and IL-4 positive cells (Th1 and Th2, respectively).ResultsSarcoidosis patients had an increased number of CD4+ -IFN-γ producing cells in IS (p = 0.003) and BALF (p = 0.01) in comparison with normal subjects. No significant differences were detected between CD4+ -IL-4 cells in BALF (p = 0.053, NS) and IS (p = 0.46, NS) between sarcoidosis patients and healthy controls. The ratio of Th1 to Th2 cells in BALF and IS was statistically different in sarcoidosis when compared with normal subjects (p = 0.007 in BALF and IS). A significant correlation was found between CD4+ IFN-γ positive cells in IS and those in BALF in sarcoidosis patients (r = 0.685, p = 0.0006).ConclusionThese data suggests that a Th1-like cytokine pattern can be observed in CD4+ T-lymphocytes in IS in patients with pulmonary sarcoidosis. Further studies are needed to explore the value of IS vs BALF in the follow-up of these patients.

Amb a 1–immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response

Background Amb a 1–immunostimulatory phosphorothioate oligonucleotide conjugate (AIC) is a novel immunotherapeutic compound consisting of purified Amb a 1 from short ragweed proteins covalently linked to an immunostimulatory phosphorothioate oligodeoxyribonucleotide. In sensitized animals AIC can stimulate an Amb a 1–specific T H1 response and decrease pulmonary reactivity to ragweed challenge. Clinical trials have documented reduced allergic response to AIC in comparison with licensed ragweed extract. Objectives We sought to determine the in vivo effect of short-course immunotherapy with AIC on eosinophilia and cytokine mRNA expression in the nasal mucosa of ragweed-sensitive patients. Methods Ragweed-sensitive patients with allergic rhinitis were treated with 6 escalating doses of AIC (0.06-12 μg, n = 28) or placebo (n = 29) at weekly intervals immediately before the 2001 ragweed season. Symptom scores and medication use were recorded for the 2001 and 2002 ragweed seasons for all patients. A subset of patients (12 receiving AIC and 7 receiving placebo) consented to have nasal biopsy specimens taken before immunization and before and after the first ragweed season. The preseason and postseason biopsy specimens were taken 24 hours after ragweed allergen challenge and compared with the initial unchallenged biopsy specimen to assess cytokine and inflammatory cell responses by using immunocytochemistry and in situ hybridization. Results AIC was safe and well tolerated by all patients. There was no difference between the AIC and placebo groups in the number of allergen-induced major basic protein–, IL-4–, IL-5–, or IFN-γ–positive cells in the mucosa in the first weeks after AIC immunization. On rechallenge and rebiopsy after the end of the 2001 ragweed season, however, AIC-treated patients had a significantly reduced increase in eosinophils and IL-4 mRNA-positive cells and an increased number of IFN-γ mRNA-positive cells compared with placebo-treated patients. No difference between treatment groups was observed in symptom scores or medication use during the first ragweed season. During the second ragweed season, however, there was a significant decrease in chest symptoms and a trend toward reduced nasal symptoms in the AIC-treated group. Conclusion Short-course immunotherapy with AIC can modify the response of nasal mucosa to allergen challenge by increasing T H1 cytokine production and decreasing T H2 cytokine production and eosinophilia. This modification was not immediate but was observed 4 to 5 months after completion of immunotherapy and seasonal ragweed-pollen exposure. The T-cell subset shift after immunization and seasonal exposure was followed by evidence of clinical efficacy in the second ragweed season without additional AIC immunizations.

Tc1 effector diversity shows dissociated expression of granzyme B and interferon-gamma in HIV infection.

To examine antigen specific cytotoxic effector T cell diversity in HIV infected individuals. We used a panel of previously defined HLA class I-restricted HIV peptides to stimulate CD8 cells in freshly isolated peripheral blood mononuclear cells of HIV infected patients, to determine cognate killing via the perforin-granzyme pathway and inflammation induced by secretion of interferon (IFN)-gamma. ELISPOT assays were used to measure the secretion of granzyme B (GzB) and IFN-gamma at single cell resolution. In all nine patients only approximately 20% of the peptides triggered a canonical Tc1 response with simultaneous release of GzB and IFN-gamma. The majority of these peptides (approximately 80%) that elicited recall responses fell into the 'single positive' category with induction of either GzB or IFN-gamma alone. The GzB positive cells did not produce interleukin (IL)-4 or IL-5. The GzB positive but IFN-gamma negative CD8 cells are programmed to induce apoptosis mediated killing without inflammation while the GzB negative and IFN-gamma positive CD8 cells could mediate inflammation without killing. This Tc1 CD8 effector cell diversity and the understanding of these differentiation mechanisms may enable the precise implementation and fine-tuning of fundamentally different defense strategies against HIV and other infections.

T cell response to respiratory syncytial virus (RSV)

Rationale Severe RSV infection in early infancy is predictive of wheezing later in life. One possible explanation for this linkage is that early childhood RSV infection causes delayed maturation of immune response that results in prolonged Th2-skewed immune response, which in turn predisposes to wheezing. Methods To explain the relationship between RSV infection and asthma, we first optimized the conditions for studying RSV specific memory T cell response by comparing three different antigen presenting cells (APCs): monocyte-derived dendritic cells (MDDCs), macrophages, and monocytes. The adult human APCs were first exposed to RSV for four hours and then cultured with autologous lymphocytes for 24 hours. Memory CD4 T cells responding to RSV were identified by intracellular staining of IFN-γ, IL-4, and IL-13, and by flow cytometry. Results Compared to macrophages and monocytes, the MDDCs increase the percentage of RSV responding IFN-γ positive cells about 9-fold. None of the APCs stimulated IL-4 or IL-13 expression in these adult cells. In addition to cytokines, we also studied T cell proliferation by 3H-thymidine uptake assay, and confirmed previous results on RSV suppression of polyclonal T cell proliferation. However, we also showed that RSV inhibits specific T cell proliferation in response to cytomegalovirus (CMV) in a dose- and time-dependent manner. Conclusions To our knowledge, this is the first demonstration of suppression of virus specific CD4 response by RSV in human system.

Restoration of tumor specific human leukocyte antigens class I-restricted cytotoxicity by dendritic cell stimulation of tumor infiltrating lymphocytes in patients with advanced ovarian cancer

Despite the large number of potentially cytotoxic tumor-infiltrating (TIL) and tumor-associated (TAL) lymphocytes accumulated in the peritoneal cavity ascitic fluid and tumor tissue, advanced ovarian cancer is a progressive disease, suggesting that TIL and TAL populations eventually become functionally suppressedin vivo. Dendritic cells (DC) are the most powerful professional antigen presenting cells known in humans and recently, ovarian tumor antigen pulsed DC have been shown to elicit tumor specific human leukocyte antigens (HLA)-class I-restricted cytotoxicity from the peripheral blood of advanced ovarian cancer patients. In this study, we have evaluated the potential of tumor antigen-pulsed fully mature DC stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced ovarian cancer patients. In addition, we have compared tumor-specific T-cell responses induced by tumor antigen-loaded DC in TIL to those induced in TAL and peripheral blood lymphocytes (PBL). DC stimulation induced powerful cytotoxicity against autologous tumor target cells in TIL-derived CD8+ T-cells from all patients tested, while autologous Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) were not lysed. Killing of autologous tumor cells was higher by CD8+ T-cells from TIL compared to PBL and TAL (P< 0.01) and was more strongly inhibited by anti-HLA class I MAb (P< 0.05 compared to PBL and TAL). Phenotypically, all cytotoxic T lymphocyte (CTL) populations were CD3+/CD8+, with variable levels of CD56 expression. Finally, although a marked Type 1 cytokine bias [ie, interferon-gamma/interleukin-4 (IFN-γhigh/IL-4low)] was observable in all DC-stimulated CD8+ T-cell populations, TIL derived CD8+ T-cells showed a higher percentage of IFN-γ positive cells compared to TAL and PBL. Taken together, these data show that tumor lysate-pulsed DC can consistently restore strong CD8+ CTL responses from TIL against autologous ovarian cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL for adoptive T-cell immunotherapy for advanced ovarian cancer.

Cytokines and adhesion molecules in duodenal mucosa of children with delayed-type food allergy.

The aim was to investigate the expression of cytokines, adhesion molecules, and activation and proliferation markers in duodenal biopsies from children with delayed-type food allergy (FA). Seven children with untreated FA (uFA), seven children with treated FA (tFA) to cow milk and/or cereals, and five normal controls furnished duodenal biopsy specimens. Additionally, five pediatric patients with celiac disease were included, serving exclusively as positive controls for in situ hybridization. Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), adhesion molecules, and activation markers were detected by immunohistochemistry, and expression of IFN-gamma and IL-4 messenger RNA was revealed by in situ hybridization. uFA patients had a higher density of IFN-gamma positive cells in the lamina propria than did tFA patients and controls (P = 0.053 and P = 0.018). Moreover, the uFA patients exhibited a higher proportion of crypt cells in mitosis than did tFA patients (P = 0.026), and stronger staining of HLA-DR in the crypts and increased density of gammadelta-T cell receptor-positive intraepithelial lymphocytes than did controls (P = 0.048 and P = 0.010). The densities of alpha(4)beta(7) positive cells in the lamina propria tended to be higher in controls than in uFA or tFA patients (P = 0.106, P = 0.073). Expression of IL-4 mRNA was significantly higher in celiac patients than in the other study groups (uFA P = 0.006, tFA P = 0.010; controls P = 0.029), and celiac patients showed higher expression of IFN-gamma mRNA than did tFA patients or controls (P = 0.017 and P = 0.016). As expected, Th1 dominance was present in the lamina propria of children with delayed-type FA. It may cause activation of epithelial cells and increase their turnover.

Immunohistochemical staining of IFN-γ positive cells in porcine reproductive and respiratory syndrome virus-infected lungs

Paraffin-embedded lungs were obtained from a previous porcine reproductive and respiratory syndrome virus (PRRSV)-challenged experiment involving three groups: an uninfected control group, a low virulence (LV, Resp PRRSV/Repro™)-infected group, and a high virulence (HV, VR-2385)-infected group. Tissues were collected at 3, 7, 10, 14 or 28 days post-inoculation (DPI) ( n=5). Lungs were examined to detect IFN-γ positive cells by immunohistochemical staining using polyclonal antibodies to IFN-γ. The microscopic lung lesions induced by the HV group were more severe than those in the LV group. A significant increase in number of lymphocytes in the HV group was observed at 10 DPI (24.90±9.79%), 14 DPI (22.00±11.47%) and 28 DPI (28.95±15.11%) ( P<0.05). A relative decrease in macrophage numbers was observed and correlated well with the increase in lymphocyte numbers when the disease progressed. IFN-γ positive cells were demonstrated in both lymphocytes and macrophages, particularly pulmonary alveolar macrophages. A significant increase in IFN-γ positive cells was found at 7 DPI (15.90±13.65%), 10 DPI (46.95±13.79%), 14 DPI (10.90±5.13%) and 28 DPI (13.40±4.89%) in the HV group ( P<0.05). The results suggested that the increase in IFN-γ positive cells in the HV group correlated well with the severity of the lung lesions, which may be because of the presence of PRRSV in the lung.

The flavonoid, quercetin, differentially regulates Th-1 (IFNγ) and Th-2 (IL4) cytokine gene expression by normal peripheral blood mononuclear cells

Flavonoids are plant metabolites that are dietary antioxidants and exert significant anti-tumor, anti-allergic, anti-inflammatory and anti-viral effects. It is generally accepted that Th-1 derived cytokines such as IL-2, IFNγ and IL-12 promote cellular immunity while Th-2 derived cytokines such as IL-4, IL-5, IL-6 exert negative immunoregulatory effects on cellular immunity while upregulating humoral immunity. The molecular mechanisms underlying the biological activities of flavonoids have not been elucidated. We hypothesize that the flavonoid, quercetin, exert significant anti-viral and anti-tumor effects possibly by modulating the production of Th-1 and Th-2 derived cytokines. Peripheral blood mononuclear cells (PBMC, 1×10 6 cells/ml) from normal subjects were cultured with different concentrations of quercetin (0.5–50 μM) for 24–72 h and supernates were quantitated for IFN-γ and IL-4 by ELISA and antiviral activity of IFNγ by bioassay. FACS analysis was done to determine the number of IFN-γ and IL-4 positive cells and RT-PCR was done to quantitate gene expression. Quercetin significantly induces the gene expression as well as the production of Th-1 derived IFNγ and the downregulates Th-2 derived IL-4 by normal PBMC. Further, quercetin treatment increased the phenotypic expression of IFNγ cells and decreased IL-4 positive cells by FACS analysis, which corroborate with protein secretion and gene expression studies. These results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of Th-1 derived cytokine, IFNγ, and inhibition of Th-2 derived cytokine, IL-4.

Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B

In murine models, overexpression of interleukin (IL)-12 and interferon (IFN)-γ can induce severe liver damage, whereas IL-10 has anti-inflammatory and hepatoprotective properties. To analyze the potential role of these cytokines in human fulminant hepatitis B, we used immunohistochemistry to study expression of IL-12, IFN-γ, and IL-10 in explant livers of 11 patients with fulminant hepatitis B, 5 patients with fulminant hepatitis due to other etiologies, 37 patients with chronic liver disease (CLD; hepatitis B virus, n = 15; hepatitis C virus, n = 10; primary biliary cirrhosis, n = 12), and 10 normal controls (NCs). Furthermore, cytokine messenger RNA (mRNA) levels were determined in the liver specimens by quantitative real-time polymerase chain reaction (PCR). In NCs, faint IL-12 expression was detected in only a few Kupffer cells, whereas sinusoidal endothelial cells, hepatic stellate cells, bile ducts, and lymphocytes expressed IL-12 in CLD and, more conspicuously, in fulminant hepatitis B. In contrast, expression of IFN-γ and IL-10 was restricted to lymphocytes and Kupffer cells, respectively. In fulminant hepatitis B, numbers of IL-12– and IFN-γ–positive cells markedly exceeded those found in CLD and NCs. A close correlation existed between IL-12 and IFN-γ expression ( r = 0.68; P < .001). In contrast, IL-10 expression was not significantly different in CLD and fulminant hepatitis. The quantitative differences in immunohistologic cytokine expression closely corresponded to the mRNA levels. In conclusion, our data indicate massive induction of the proinflammatory cytokines IL-12 and IFN-γ in fulminant hepatitis B, which is apparently not counterbalanced by the anti-inflammatory cytokine IL-10. This cytokine imbalance may play an important role in promoting inflammatory reactions leading to massive liver damage in fulminant hepatitis B. (H EPATOLOGY 2002;36:1001-1008.)

Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B

In murine models, overexpression of interleukin (IL)-12 and interferon (IFN)-γ can induce severe liver damage, whereas IL-10 has anti-inflammatory and hepatoprotective properties. To analyze the potential role of these cytokines in human fulminant hepatitis B, we used immunohistochemistry to study expression of IL-12, IFN-γ, and IL-10 in explant livers of 11 patients with fulminant hepatitis B, 5 patients with fulminant hepatitis due to other etiologies, 37 patients with chronic liver disease (CLD; hepatitis B virus, n = 15; hepatitis C virus, n = 10; primary biliary cirrhosis, n = 12), and 10 normal controls (NCs). Furthermore, cytokine messenger RNA (mRNA) levels were determined in the liver specimens by quantitative real-time polymerase chain reaction (PCR). In NCs, faint IL-12 expression was detected in only a few Kupffer cells, whereas sinusoidal endothelial cells, hepatic stellate cells, bile ducts, and lymphocytes expressed IL-12 in CLD and, more conspicuously, in fulminant hepatitis B. In contrast, expression of IFN-γ and IL-10 was restricted to lymphocytes and Kupffer cells, respectively. In fulminant hepatitis B, numbers of IL-12– and IFN-γ–positive cells markedly exceeded those found in CLD and NCs. A close correlation existed between IL-12 and IFN-γ expression (r = 0.68; P < .001). In contrast, IL-10 expression was not significantly different in CLD and fulminant hepatitis. The quantitative differences in immunohistologic cytokine expression closely corresponded to the mRNA levels. In conclusion, our data indicate massive induction of the proinflammatory cytokines IL-12 and IFN-γ in fulminant hepatitis B, which is apparently not counterbalanced by the anti-inflammatory cytokine IL-10. This cytokine imbalance may play an important role in promoting inflammatory reactions leading to massive liver damage in fulminant hepatitis B. (HEPATOLOGY 2002;36:1001-1008.)

Interferon-gamma, Interleukin-18, Monokine Induced by Interferon-gamma and Interferon-gamma-inducible Protein-10 in Histiocytic Necrotizing Lymphadenitis

Apoptosis of histiocytes is a characteristic feature of necrotizing lymphadenitis (HNL). Recent studies have indicated that Fas and perforin-based pathways are involved in the apoptotic process of HNL. Elevated levels of serum interferon (IFN)-γ are reported in HNL. The CXC chemokine interferon-γ-inducible protein-10 (IP-10) and monokine induced by interferon-γ (MIG) cause tissue necrosis, and interleukin (IL)-18 induces the expression of IFN-γ and Fas ligand (FasL) by T and natural killer (NK) cells. This study was designed to determine the expression of IFN-γ, IL-18, MIG and IP-10 in HNL. Ten cases of HNL were analyzed by using immunohistochemical staining and/or reverse transcriptase–polymerase chain reaction (RT-PCR). As a control, we included four cases of non-specific lymphadenitis. MIG and IP-10 proteins, which enhance the release of granzyme, showed a similar distribution pattern in viable tissues surrounding dead tissue, mostly within histiocytes, and lymphocytes in HNL. IL-18 was located within histiocytes, especially phagocytic histiocytes, but not within lymphocytes. In addition, IFN-γ-positive lymphocytes were frequently detected in the surrounding dead tissue, and the lymphocytes in the same area were frequently positive for CXCR3, a specific receptor of MIG and IP-10. In non-specific lymphadenitis, MIG, IP-10 and IL-18 positive cells were detected, but their numbers were relatively small compared with HNL, while IFN-γ positive cells were rarely encountered. Our findings suggest that the cytokine and chemokine pathways of IFN-γ, IL-18, MIG and IP-10 play an important role in the pathogenesis of apoptosis associated with HNL.

Measurement of intracellular interferon-gamma and interleukin-4 in whole blood T lymphocytes from patients with systemic lupus erythematosus

Contradictory data are available about the dominance of T-helper 1 (T H1), or T-helper 2 (T H2) cytokines in systemic lupus erythematosus (SLE). Therefore, intracellular interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production of T lymphocytes was measured in whole blood of healthy donors and active and inactive SLE patients by flow cytometry. The percentage of IFN-γ and IL-4 positive cells was low (<1%) in unstimulated samples of the healthy controls, while that of IFN-γ and IL-4 positive cells in the stimulated cells was 25.2±10.6% and 0.6±1.5%, respectively. No significant difference was found between SLE patients and healthy controls and between active and inactive patients in these parameters either in the unstimulatad or in the stimulated samples. One patient with severe disease had as high as 11.8% IL-4 positive cells and 12.5% IFN-γ positive cells in the stimulated samples, but after the initiation of intensive corticosteroid and cytostatic therapy, the percentage of IL-4 positive T cells decreased (4.76%) while that of IFN-γ positive T cells increased (47.91%). We conclude that the intracellular IL-4 and IFN-γ expression of T lymphocytes does not differ markedly between SLE patients and healthy controls, with the possible exception of severe disease, when marked IL-4 overproduction may exist beside low IFN-γ production. Furthermore, corticosteroid and cytostatic therapy might normalize this altered IFN-γ/IL-4 ratio.

CONSTITUTIVELY DECREASED TH3 SUPPRESSOR CELLS MAY ALLOW LOW-DOSE SENSITISATION IN CMSE

82 Aims: Many infants sensitise to dietary antigens despite exclusive breast feeding. While acknowledged an increasingly common cause of cows' milk sensitive enteropathy (CMSE), multiple food intolerances are now recognized 1,2. A defect in oral tolerance in atopic infants, specifically for low dose antigen and due to impaired generation of TH3 cells (TGF-β secreting T cells) has been postulated as a potential cause 3. We have thus studied mucosal TH1, TH2 and TH3 cells in CMSE and other enteropathies. Methods: Spontaneous production of IL-2, IFN-γ (TH1), IL-4 (TH2) and TGF-β (TH3) was analysed by flow cytometry (FACS) in the intraepithelial (IEL) and lamina propria (LPL) lymphocytes from 54 children with normal histology (n=17), active (16) and treated (9) CMSE, coeliac disease (7) or inflammatory enteropathy (5). Results: In contrast to coeliac disease and inflammatory enteropathy, active CMSE was characterised by reduced TH1 cytokine production. CD4+ IEL showed reduced IFNγ and TGFβ in CMSE at diagnosis, not seen on cows' milk exclusion. CD4+ LPL showed reduced IL-2 and IFNγ positive cells, again only in active disease. CD8+ IEL's and LPL's showed similar reduction of TH1 cytokines in active CMSE. However a striking decrease in TGFβ+ CD8+ IELs and LPLs was also seen, not only in active disease but persisting on cow's milk exclusion. Conclusions: We have found evidence of reduced TGFβ production by mucosal CD8+ cells in active CMSE and following cow's milk exclusion. CD4+ cells also show reduced TGFβ production, but only in active disease. Impaired generation of TH3 cells may thus be a predisposing factor for the development of CMSE. These finding are consistent with the hypothesis of impaired low-dose oral tolerance in atopic infants 3.