Cytokine expression in murine cytomegalovirus-induced myocarditis: modulation with interferon-alpha therapy

Cytomegalovirus-induced myocarditis is largely immune-mediated. BALB/c mice produced higher levels of IL-4 in the heart indicative of a Th2-like response. Although IL-6, IL-10, IL-18, and TNF-alpha were produced in the heart during acute infection, BALB/c mice lacked a substantial IL-2 and IFN-gamma response. Conversely, C57BL/6 mice produced significant levels of IFN-gamma in the heart with no significant levels of IL-4 or IL-6, suggestive of a dominant Th1-like response to virus infection. IFN-alpha/beta immunotherapy is known to suppress the development of MCMV-myocarditis. Cytokine secretion in IFN-stimulated MCMV-infected BALB/c myocytes was found to be IFN subtype-dependent with elevation of IL-6 and IL-18 levels. During the chronic phase of disease, IFNA6 DNA treatment in vivo increased IL-18 production in the heart. These results suggest that IFN subtype therapy may have immunomodulating effects in reducing disease severity in BALB/c mice via regulation of cytokine production in the heart.     

Dynamics of the cytokine messenger RNA expression pattern in the liver of baboons infected with Schistosoma mansoni

Periovular granulomas are the major lesions in baboons infected with Schistosoma mansoni. Temporal Northern blot analysis of cytokine messenger RNA (mRNA) expression in granulomatous baboon livers demonstrated tissue-specific expression. Interleukin 1 beta (IL-1beta, IL-6, tumor necrosis factor alpha, and migration inhibitory factor (MIF) mRNAs were expressed strongly at week 6 of infection and decayed thereafter, whereas interferon gamma (IFN-gamma), IL-2, IL-10, and IL-12 mRNAs were first expressed at week 12, with IFN-gamma and IL-12 mRNA expression persisting until week 17. IL-4 and IL-5 mRNAs first appeared at week 12, with IL-4 persisting unchanged and IL-5 increasing by week 17. Thus, egg deposition induced strong hepatic expression of proinflammatory and downregulatory cytokines. The cooccurrence of IL-2, IFN-gamma, IL-4, and IL-5 mRNAs at week 12 confirms that baboons, like humans, show a mixed type 1-type 2 cytokine response. When granulomas had become smaller at 17 wk, IFN-gamma, IL-4, and IL-5 were the only cytokine mRNAs that were expressed strongly, implicating them in granuloma modulation. The early expression of MIF mRNA and MIF's role as the main counterregulator of glucocorticoid immunosuppression ties in with our earlier demonstrations of circulating adrenal steroids changing with the progression of schistosomiasis in baboons and of proinflammatory cytokine mRNA expression in the hypothalamic-pituitary-adrenal axis tissues of infected baboons. Together, these data imply neuroendocrinological influences on disease progression in schistosomiasis.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.     

Scorpion venom increases mRNA expression of lung cytokines

Previous studies have demonstrated that scorpion toxins increase the serum levels of IL-1, IL-6, INF-gamma, and GM-CSF in patients with severe shock and pulmonary edema. Moreover, it has been shown that experimental models of scorpion envenomation presented an increase in serum levels of IL-1, IL-6, IFN-gamma and nitric oxide. Thus, it is possible that the cytokine release may contribute to the onset and maintenance of the pulmonary edema induced by scorpion venom. This study was designed to investigate whether inflammatory and non-inflammatory cytokines, contribute to the pulmonary injury induced by infusion of Tityus serrulatus scorpion toxin in rats. We show that scorpion venom not only increases the expression of mRNA pulmonary inflammatory cytokines but also non-inflammatory cytokines as well. Moreover, the expression of IL-1alpha, IL-1beta and IL-6 mRNA was shown to be higher among the remaining detectable cytokines. The findings of this study provide additional insight towards the understanding of the pathophysiology of the pulmonary edema induced by scorpion venom. The increased level of pulmonary cytokines observed during the pulmonary edema may be responsible for the exacerbation and maintenance of the inflammatory response to scorpion venom in the lungs.     

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.     

In vivo molecular analysis of lymphokines involved in the murine immune response during Schistosoma mansoni infection. II. Quantification of IL-4 mRNA, IFN-gamma mRNA, and IL-2 mRNA levels in the granulomatous livers, mesenteric lymph nodes, and spleens during the course of modulation.

Parasite egg-induced granulomas are the primary pathogenic lesions in murine schistosomiasis mansoni. This cell-mediated granulomatous response is specific for soluble egg Ag and appears to be mediated predominantly by CD4+ Th2 cells. As infection progresses from the acute to the chronic phase, the cell-mediated anti-soluble egg Ag responses attenuate in a process termed modulation. In this study the hypothesis that modulation is effected by a chronic phase increase in Th2-inhibiting Th1 cell activity was investigated. Northern blot quantification of mRNA specific for the Th2 lymphokine, IL-4, and the Th1 lymphokines, IFN-gamma and IL-2, in the spleens, mesenteric lymph nodes, and granulomatous livers of mice infected for various lengths of time over the course of modulation was performed. Also, the capacity of mitogen- and Ag-stimulated spleen cells to produce message for these lymphokines was compared. Peak tissue levels of both IL-4 mRNA and IFN-gamma mRNA were seen in acutely infected mice, and levels of both messages declined as infection became chronic. Stimulated spleen cells from acutely infected mice also produced higher levels of IL-4 and IFN-gamma mRNA than cells from chronically infected mice. IL-2 mRNA was never detected in any tissue sample but was detected in the stimulated spleen cells, again with acute phase levels higher than chronic phase levels. Hence, this study shows no evidence for increased Th1 cell activity during chronic infection and suggests that modulation may be effected by a generalized suppression of lymphokine synthesis.     

Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.     

Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.     

Cytokine levels (IL-6 and IFN-gamma), acute phase response and nutritional status as prognostic factors in lung cancer

Raised serum levels of interleukin 6 (IL-6) have been described in cancer patients. This cytokine mediates the acute phase response and has been also involved in the pathogenesis of cancer cachexia. The objectives of the present study were: (1) to determine the relationships of IL-6 and other cytokines with neoplasia extension, acute phase response and nutritional status, in lung cancer patients; and (2) to establish the prognostic value of serum cytokine levels. A prospective study in which IL-1, IL-2, IL-6, tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) have been determined in 66 newly diagnosed lung cancer patients. Nutritional status was assessed objectively. Serum levels of growth hormone (GH), insulin growth factor 1 (IGF-1) and acute phase reactants as C Reactive Protein, alpha1 antitrypsin and ferritine, were determined. Increased IL-6 levels were related to extensive disease, impaired performance status, enhanced acute phase response and malnutrition. Raised serum IL-6 levels, extensive disease, low Karnofsky index, malnutrition, acute phase response and low IFN-gamma were all related to a shorter survival. When assessed by a multivariate analysis, IL-6 kept its independent prognostic value together with age, disease extension, and decreased IFN-gamma serum levels.IL-6 is increased in lung cancer patients, enhances the acute phase response in them, and is correlated with poor nutritional status, impaired performance status and shorter survival.     

Experimental autoimmune encephalomyelitis on the SJL mouse: effect of gamma delta T cell depletion on chemokine and chemokine receptor expression in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that is a model for multiple sclerosis. Previously, we showed that depletion of gamma delta T cells significantly reduced clinical and pathological signs of disease, which was associated with reduced expression of IL-1 beta, IL-6, TNF-alpha, and lymphotoxin at disease onset and a more persistent reduction in IFN-gamma. In this study, we analyzed the effect of gamma delta T cell depletion on chemokine and chemokine receptor expression. In the CNS of control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, MIP-2, inducible protein-10, and monocyte chemoattractant protein-1 were detected at disease onset, increased as disease progressed, and fell as clinical signs improved. In gamma delta T cell-depleted animals, all of the chemokine mRNAs were reduced at disease onset; but at the height of disease, expression was variable and showed no differences from control animals. mRNA levels then fell in parallel with control EAE mice. ELISA data confirmed reduced expression of MIP-1 alpha and monocyte chemoattractant protein-1 at disease onset in gamma delta T cell-depleted mice, and total T cell numbers were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were observed, and these were elevated in EAE animals. mRNAs for CCR2 were also detected in the CNS of affected mice. Depletion of gamma delta T cells reduced expression of CCR1 and CCR5 at disease onset only. We conclude that gamma delta T cells contribute to the development of EAE by promoting an inflammatory environment that serves to accelerate the inflammatory process in the CNS.     

IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.     

Peak protein expression of IL-2 and IFN-gamma correlate with the peak rejection episode in a spontaneously tolerant model of rat liver transplantation

Spontaneous orthotopic liver transplant (OLT) tolerance occurs uniformly between the inbred rat strains of DA (MHC haplotype RT1(a)) into PVG (RT1(c)) despite a fully allogeneic barrier. Animals transplanted in this combination do, however, undergo a rejection episode which appears to be self-limiting. In order to clarify this further we under took in situ measurements of the cytokines IL-2, IFN-gamma and TNF-alpha prior to, during and post rejection episode. The cytokine protein product was examined via immunoblotting assays and mRNA levels by RT-PCR. Comparisons were also made for syngeneic transplant combinations over the same time period. Peak protein expression of IL-2 and, to a lesser extent, IFN-gamma, occurred during the rejection episode between days 10 and 14. IFN-gamma was still present in syngeneic OLT on day 10 but was only present in allogeneic OLT on day 14. IL-2 was only detectable in allogeneic OLT on days 10 and 14. Transient increases in TNF-alpha occurred in allogeneic and syngeneic OLT with TNF-alpha levels falling by the peak rejection episode. Immunoblotting also confirmed the ability of hepatocytes to produce each of the cytokines studied. mRNA levels, by contrast, were maximal at days 1 and 10 for IL-2 and day 3 for IFN-gamma in allogeneic OLT when compared with syngeneic and non-transplanted controls. Earlier increases in IL-2 and IFN-gamma mRNA and time of peak protein expression do not correlate in this model. We therefore conclude that the measurement of peak mRNA levels alone are not enough to evaluate the rejection process especially since it is the cytokine protein products which have potential biological activity.     

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-gamma as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi

We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.     

Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomas

The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of Th1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin lymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-gamma, IL-10, IL-4, TGF-beta) were determined by real-time PCR technique. Comparing to the control group, we found lower expression of mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of impaired immunity in these patients.     

Expression and regulation of histidine decarboxylase mRNA expression in the uterus during pregnancy in the mouse

It has been hypothesized that hormonally regulated histamine production plays a role in preparation of the uterus for implantation. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production. The current study was designed to determine intrauterine expression of HDC mRNA expression during pregnancy in the mouse. High levels of HDC mRNA expression were observed in the preimplantation mouse uterus with peak expression occurring on day 4. High levels of HDC mRNA expression were also detected in the post-implantation uterus. In an effort to determine whether HDC mRNA is regulated by pro-inflammatory cytokines, the HDC mRNA pattern was compared to intrauterine expression of mRNA's for interleukin-1alpha (IL-1alpha), IL-1beta, macrophage chemotactic protein-1 (MCP-1) and RANTES (regulated on activation, normal T expressed and secreted) during the peri-implantation period. IL-1beta, MCP-1 and RANTES mRNA levels were increased in the uterus on days 1-2 and on days 4-5. Increased expression of IL-1alpha mRNA was observed on days 1-2 and days 5-7. There was no clear relationship between HDC mRNA expression and cytokine/chemokine mRNA expression. Progesterone-stimulated intrauterine expression of HDC mRNA. Intrauterine cytokine/chemokine mRNA was also hormonally regulated. This data allowed the possibility that one or more of these pro-inflammatory cytokines could be involved in regulating intrauterine HDC mRNA production. Recombinant IL-1alpha, IL-1beta, MCP-1 and RANTES all failed to induce HDC mRNA expression in the preimplantation uterus in a mouse pseudopregnancy model. At the same time, IL-1beta induced the expression of mRNA for each of the four cytokines/chemokines. Despite the fact that these were also produced in the uterus during pregnancy and were hormonally regulated, none of these cytokines induced intrauterine HDC mRNA expression. The data suggest that progesterone is involved in the regulation of HDC mRNA expression in the preimplantation uterus, but IL-1alpha/beta, MCP-1 and RANTES, which have been reported to regulate histamine synthesis during inflammatory processes, do not appear to play a role.     

Anti-CD3 antibody induces rapid expression of cytokine genes in vivo

Anti-CD3 antibody was administered to mice i.v. and the kinetics of spleen cell cytokine mRNA expression studied by Northern analysis. Untreated mice and mice receiving control antibody had low or undetectable amounts of mRNA for c-fos, c-myc, IL-2, IL-4, and IFN-gamma. After injection of anti-CD3 antibody, substantial increases in all were found. Induction of c-fos was detected at 10 min and of c-myc at 30 min after injection. IL-2, IL-4, and IFN-gamma mRNA were induced by 30 min and reached peak levels at 60 min. Thereafter, IL-2 and IL-4 mRNA declined, whereas IFN-gamma mRNA persisted. The induced cytokine mRNA was not observed in athymic nu/nu mice nor in normal spleen cells from which T cells had been depleted in vitro. The early in vivo induction of IL-4 mRNA contrasts with prior in vitro studies in which IL-4 production was difficult to detect after primary stimulation. To assess the possibility that many T cells had been preprimed in vivo, germfree mice were compared with conventional mice and no differences in cytokine mRNA were found. These data show that T cell-dependent IL message production can be induced rapidly in vivo without prepriming and that the cytokine messages induced after anti-CD3 antibody administration do not suggest a predominance of either Th1 or Th2 type cells.     

Cytokine gene activation in synovial membrane, regional lymph nodes, and spleen during the course of rat adjuvant arthritis.

Cytokine gene activation was assessed during rat adjuvant arthritis (AA) in synovial membrane (SM), popliteal lymph node (popl-LN), and spleen, using semiquantitative, competitive RT-PCR. Changes in the popl-LN were considerably higher than in spleen or SM. In the preclinical phase (day 6), cytokine mRNA elevations occurred exclusively in the popl-LN and included IFN-gamma, IL-1beta, IL-5, IL-6, and IL-10. In the acute phase (days 13-16) all three organs became involved: (i) in the SM, significant elevations were limited to IL-1beta and IL-6, which, notably, correlated positively with the degree of arthritis; (ii) in the popl-LN, IFN-gamma, IL-1beta, IL-6, and IL-10 (but not IL-5) were still elevated, while IL-2 rose significantly; (iii) in the spleen, TNF-alpha peaked simultaneously with the arthritis score (day 16) and dramatically dropped thereafter. Upon transition into the chronic phase (day 20) the following phenomena were observed: (i) IL-1beta and IL-6 were still significantly increased in the SM; (ii) IFN-gamma, IL-1beta, IL-2, IL-6, and IL-10 were still elevated in the popl-LN; and (iii) there was a progressive rise of IL-5 mRNA in the spleen, positively correlated with the arthritis score. In conclusion, cytokines with pro- and anti-inflammatory functions overlap throughout disease, but in different organ-related patterns. Local (SM) and regional (popl-LN) IL-1beta and IL-6, elevated throughout the entire course of AA, may directly contribute to disease severity. While in AA spleen TNF-alpha appears to be a systemic marker of acute disease, spleen IL-5 may be involved in disease resolution.