Thalidomide alleviates neuropathic pain through microglial IL-10/ß-endorphin signaling pathway.

Thalidomide is an antiinflammatory, antiangiogenic and immunomodulatory agent which has been used for the treatment of erythema nodosum leprosum and multiple myeloma. It has also been employed in treating complex regional pain syndromes. The current study aimed to reveal the molecular mechanisms underlying thalidomide-induced pain antihypersensitive effects in neuropathic pain. Thalidomide gavage, but not its more potent analogs lenalidomide and pomalidomide, inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain rats induced by tight ligation of spinal nerves, with ED50 values of 44.9 and 23.5 mg/kg, and Emax values of 74% and 84% MPE respectively. Intrathecal injection of thalidomide also inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain. Treatment with thalidomide, lenalidomide and pomalidomide reduced peripheral nerve injury-induced proinflammatory cytokines (TNFα, IL-1ß and IL-6) in the ipsilateral spinal cords of neuropathic rats and LPS-treated primary microglial cells. In contrast, treatment with thalidomide, but not lenalidomide or pomalidomide, stimulated spinal expressions of IL-10 and ß-endorphin in neuropathic rats. Particularly, thalidomide specifically stimulated IL-10 and ß-endorphin expressions in microglia but not astrocytes or neurons. Furthermore, pretreatment with the IL-10 antibody blocked upregulation of ß-endorphin in neuropathic rats and cultured microglial cells, whereas it did not restore thalidomide-induced downregulation of proinflammatory cytokine expression. Importantly, pretreatment with intrathecal injection of the microglial metabolic inhibitor minocycline, IL-10 antibody, ß-endorphin antiserum, and preferred or selective µ-opioid receptor antagonist naloxone or CTAP entirely blocked thalidomide gavage-induced mechanical antiallodynia. Our results demonstrate that thalidomide, but not lenalidomide or pomalidomide, alleviates neuropathic pain, which is mediated by upregulation of spinal microglial IL-10/ß-endorphin expression, rather than downregulation of TNFα expression.

Association of IL-10 Gene Polymorphism With IL-10 Secretion by CD4 and T Regulatory Cells in Human Leprosy.

Leprosy is a chronic bacterial disease caused by Mycobacterium leprae. Cytokines are known to play vital role as a peacekeeper during inflammatory and other immunocompromised conditions such as leprosy. This study has tried to bridge the gap of information on cytokine gene polymorphisms and its potential role in the pathogenesis of leprosy. Interleukin-10 (IL-10) is an immunosuppressive cytokine, found to be elevated in leprosy that accounted for the suppression of host's immune system by regulating the functions of other immune cells. T helper cells and T regulatory (Tregs) cells are the major source of IL-10 in lepromatous leprosy patients. In this study, we have documented the association of IL-10 cytokine gene polymorphism with the disease progression. A total of 132 lepromatous leprosy patients and 120 healthy controls were analyzed for IL-10 cytokine gene polymorphisms using PCR-SSP assay and flow cytometry was used to analyze IL-10 secretion by CD4 and Tregs in various genotype of leprosy patients. The frequencies of IL-10 (-819) TT and IL-10 (-1082) GG genotypes were significantly higher in leprosy patients as compared to healthy controls. This observation advocates that these genotypes were associated with the susceptibility and development of the disease. In addition, flow cytometry analysis demonstrated an increased number of IL-10 producing CD4 and Treg cells in IL-10 (819) TT genotype compared to CT and CC genotypes. These observations were further supported by immunohistochemical studies. Therefore, we can conclude that IL-10 cytokine gene polymorphisms by affecting its production can determine the predilection and progression of leprosy in the study population.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.

Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy.

Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.

IL-10 gene promoter polymorphisms and leprosy in a Colombian population sample.

INTRODUCTION: Polymorphisms in promoters of genes code for cytokines that affect transcription levels. Several have been associated with leprosy patients that have functional and clinical implications. OBJECTIVE: Polymorphisms in the promoter of the IL10 gene of leprosy patients will be compared frequencies in normal population. MATERIALS AND METHODS: SNPs (single nucleotide polymorphism) -1082 A/G (rs1800896), -819C/T (rs1800871), and -592A/C (rs1800872) were identified in 100 leprosy patients and in a control group of 100 volunteers from a leprosy endemic region of Colombia. RESULTS: The genotypes C/C and C/T in the SNP -819 were associated together with leprosy (OR=4.34, p<0.001).Similarly, the genotypes C/C and C/A in the -592 SNP showed an association (OR=4.3, p<0.001). The haplotypes -819C-519C and -1082A-819C-592C showed significant association (OR=4.34, p<0.001 and OR=6.25, p<0.001) respectively. These haplotypes in homozygosis conditions were also associated with leprosy: -819C-519C/-819C-519C (OR=4.34, p<0.001), -1082A -819C-592C/-1082A -819C-592C (OR=1.90, p=0.04). The SNP -1082 was not associated with leprosy in this population. CONCLUSIONS: The haplotypes associated with leprosy, -1082A-819C-592C/-1082A-819C-592C, have been reported as low producers of IL-10. Functionally, the low production of IL-10 may have immune response consequences and clinical implications. Additional haplotypes of IL-10 have been reported as markers for leprosy susceptibility or resistance in other ethnic populations. This suggests that differences in distribution of diverse IL-10 gene polymorphisms among ethnic groups may indicate important gene-disease associations.

Genetic, epidemiological and biological analysis of interleukin-10 promoter single-nucleotide polymorphisms suggests a definitive role for -819C/T in leprosy susceptibility.

Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-alpha (LTA) and vitamin-D receptor (VDR). Here, we combined a case-control study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the -819T allele is associated with leprosy susceptibility either in the case-control or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the -819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that -819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy.

[History for animal model of Hansen's disease and characteristics of leprosy in hypertensive nude rat].

The long search of an animal model for leprosy were carried out as many researchers since the Mycobacterium leprae discovery by Dr. Hansen in 1874. The remarkable results were left after the development of the foot-pad method by Dr. Shepard in 1960. The introduction of the T-R mouse and athymic (nude) mouse for leprosy research, alsospontaneity examples of Hansen's disease was reported to armadillo, chimpanzee and mangabay monkey, and it was confirmed that Hansen's disease was the zoonosis. Although, We have established a congenic hypertensive nude rat, SHR/NCrj-rnu (SHR.F344-Foxn(rnu)), carrying nude (rnu) and hypertension genes. SHR/NCrj-rnu rats obtained showed high susceptibility to M. leprae and showed a characteristic disease with a progressive pattern of leproma formation. Also this hypertensive nude rat strain produce high level of IL-10. Therefore, congenic hypertensive nude rat may be useful for an animal model to leprosy.

C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kappaB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kappaB subunit p65, but only after TLR-induced activation of NF-kappaB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylation-dependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens.

Contribution of GM-CSF on the enhancement of the T cell-stimulating activity of macrophages.

Mycobacterium leprae is an intracellular parasitic organism that multiplies in macrophages (MØ). It inhibits the fusion of mycobacterial phagosome with lysosome and induces interleukin (IL)-10 production from macrophages. However, macrophages are heterogenous in various aspects. We examined macrophages that differentiated from monocytes using either recombinant (r) granulocyte-MØ colony-stimulating factor (GM-CSF) (these MØ are named as GM-MØ) or rMØ colony-stimulating factor (M-CSF) (cells named as M-MØ) in terms of the T cell-stimulating activity. Although both macrophages phagocytosed the mycobacteria equally, GM-MØ infected with M. leprae and subsequently treated with IFN-gamma- and CD40 ligand (L) stimulated T cells to produce interferon-gamma (IFN-gamma), but M-MØ lacked the ability to stimulate T cells. While M-MØ mounted a massive IL-10 production, GM-MØ did not produce the cytokine on infection with M. leprae. M. leprae-infected, IFN-gamma- and CD40L-treated GM-MØ expressed a higher level of HLA-DR and CD86 Ags than those of M-MØ, and expressed one of the dominant antigenic molecules of M. leprae, Major Membrane Protein-II on their surface. These results indicate that GM-CSF, but not M-CSF, contributes to the up-regulation of the T cell-stimulating activity of M. leprae-infected macrophages.

Effects of prednisolone treatment on cytokine expression in patients with leprosy type 1 reactions.

Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-10, and transforming growth factor beta1 (TGF-beta1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-alpha and IL-10 in skin lesions. Increased production of TGF-beta1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-alpha, IL-1beta, and TGF-beta1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis.

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections.

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Differential production of interleukin-10 and interleukin-12 in mononuclear cells from leprosy patients with a Toll-like receptor 2 mutation.

Toll-like receptor 2 (TLR2) is a key mediator of the immune response to mycobacterial infections, and mutations in TLR2 have been shown to confer susceptibility to infection with mycobacteria. This study investigated the profiles of cytokines, such as interferon (IFN)-gamma, interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-alpha in response to Mycobacterium leprae in peripheral blood mononuclear cells (PBMC) with the TLR2 mutation Arg677Trp, a recently reported polymorphism that is associated with lepromatous leprosy. In leprosy patients with the TLR2 mutation, production of IL-2, IL-12, IFN-gamma, and TNF-alpha by M. leprae-stimulated PBMC were significantly decreased compared with that in groups with wild-type TLR2. However, the cells from patients with the TLR2 mutation showed significantly increased production of IL-10. There was no significant difference in IL-4 production between the mutant and wild-type during stimulation. Thus, these results suggest that the TLR2 signal pathway plays a critical role in the alteration of cytokine profiles in PBMC from leprosy patients and the TLR2 mutation Arg677Trp provides a mechanism for the poor cellular immune response associated with lepromatous leprosy.

Differential production of systemic and intralesional gamma interferon and interleukin-10 in nodular and ulcerative forms of Buruli disease.

Buruli disease, caused by Mycobacterium ulcerans, is the third most important mycobacterial disease in humans besides tuberculosis and leprosy. We have compared systemic and intralesional cytokine production in patients presenting with a nodular form and a necrotizing, ulcerative form of the disease. Gamma interferon (IFN-gamma) levels in response to whole M. ulcerans and Mycobacterium bovis BCG bacilli and in response to purified Ag85 protein from BCG were lower in peripheral blood mononuclear cells (PBMC) cultures from Buruli disease patients than in PBMC from healthy purified protein derivative-positive contacts. Interleukin-4 (IL-4) and IL-13 content was below the detection threshold in these PBMC cultures. IFN-gamma production after stimulation with M. ulcerans was significantly lower (P < 0.05) in PBMC cultures from patients with ulcers than in those from patients with nodules. On the other hand, PBMC from Buruli disease patients produced significant levels of IL-10 in response to M. ulcerans (but not to M. bovis BCG) and production was highest in patients with the ulcerative form. Third, semiquantitative reverse transcription-PCR analysis demonstrated a similar difference in the local, intralesional cytokine profile for the two forms of the disease: high IFN-gamma but low IL-10 mRNA levels in nodular lesions and high IL-10 but low IFN-gamma mRNA levels in ulcerative lesions. Intralesional IL-4 and IL-13 mRNA levels were low and only detected in patients with the ulcerative form. Our results indicate, although they do not formally prove, that production of IL-10 rather than production of IL-4 or IL-13 by Th2-type T cells may be involved in the low M. ulcerans-specific IFN-gamma response in Buruli disease patients.

Immunological cytokine correlates of protective immunity and pathogenesis in leprosy.

The in vitro production of interferon (IFN)-gamma, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and IL-10 by blood mononuclear cells in response to whole Mycobacterium leprae and polyclonal stimulii of 23 individuals, representing a variety of conditions in relation to exposure/susceptibility to M. leprae, was assayed. In most cases, healthy household contacts of newly diagnosed multibacillary leprosy patients, designated exposed household contacts (EC), showed low-to-undetectable in vitro IFN-gamma production in addition to substantial TNF-alpha production in response to M. leprae. In contrast, peripheral blood mononuclear cells from previously exposed contacts (R) regarded as resistant-to-leprosy released low-to-moderate levels of IFN-gamma together with a mixed cytokine profile resembling a T helper (Th)0-type response. TNF-alpha/IL-10 ratios in response to M. leprae and Concanavalin A were significantly higher in EC than in R contacts suggesting a role for the TNF-alpha/IL-10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine profiles of leprosy patients were taken as reference points. Post-treatment lepromatous leprosy patients secreted relatively high levels of IL-10 in response to M. leprae, whereas one self-cured tuberculoid leprosy patient produced simultaneously high levels of IFN-gamma and TNF-alpha. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette-Guérin (BCG) vaccination were investigated. Vaccination induced amplification of IFN-gamma production with a concomitant decrease in TNF-alpha/IL-10 ratios that resembled the cytokine pattern observed in R contacts. IFN-gamma production was observed in response to both a cross-reactive antigen (Ag 85) and a M. leprae-specific protein (MMP-I), which attests to a BCG nonspecific stimulation of the immune system, thereby casting these antigens as likely candidates for inclusion in a subunit vaccine against leprosy. Finally, a model for protective x pathologic response to mycobacteria is presented.

Serum IL-1ra is elevated in lepromatous leprosy patients.

Patterns of production of specific cytokines are accepted as standards for T-lymphocyte subsets in diseases caused by intracellular parasites. These lymphocyte subsets (Th1 and Th2) have been associated with the different poles of the leprosy spectrum. Lepromatous leprosy (LL) onset correlates with cytokines produced by Th2 cells on the grounds of the patient's poor cellular immune response, i.e., interleukin 2 (IL-2) and gamma interferon (IFN-gamma) deficiency. On the other hand, tuberculoid leprosy (TL) has been associated with a Th1 response. Moreover, pro-inflammatory cytokines like IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) play a major role in chronic inflammatory pathologies being IL-1ra and TNF-alpha soluble receptors, natural counterbalancing inhibitors. In light of this background, we decided to measure serum levels of IL-1 beta, IL-1ra, TNF-alpha and IL-6 in LL and TL patients, and we also studied the production in vitro of Th1 (IFN-gamma, IL-2), Th2 (IL-4, IL-10) and TNF-alpha cytokines. Our data showed that IL-1ra is highly elevated in sera from LL patients; there were no differences in Th2 cytokine levels and there were diminished levels in Th1 cytokines.

Lepromatous leprosy patients show T helper 1-like cytokine profile with differential expression of interleukin-10 during type 1 and 2 reactions.

Some leprosy patients suffer from clinical episodes associated with tissue damage which are designated as Type 1 (reversal reaction) when localized to the lesions and Type 2 (erythema nodosum leprosum, ENL) when accompanied by systemic involvement. We had reported earlier that stable, non-reaction lepromatous leprosy subjects show T helper 2 (Th2)- and Th0- but not Th1-like responses in the peripheral blood. To further understand the development of Th-like responses during disease, 32 lepromatous patients undergoing reactions were studied using cytokine-specific reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in peripheral blood and some skin biopsies. Of interest was the evidence of a Th1-like response with presence of interferon-gamma (IFN-gamma) and absence of interleukin-4 (IL-4) mRNA in the peripheral blood mononuclear cells (PBMC) of 85 and 64% of Type 1 and 2 reaction patients, respectively, and in all reaction sites. Whereas a Th0- was seen in some, a Th2-like response was absent. IL-12p40 mRNA was seen in 21/25 ENL and all Type 1 reaction subjects irrespective of the Th phenotype. IL-12p40 and IFN-gamma were detectable in unstimulated PBMC suggesting an in vivo priming during reactions. IL-10 was mainly associated with adherent cells and showed a differential expression in the two reactions. It was present in the PBMC of ENL but not in reversal reaction patients. Moreover, it was not detectable in the skin lesions of either type of reactions. A Th1-like cytokine profile was associated with immunopathology and persisted up to 6-7 months after the onset of reactions.

Interferon-gamma differentially regulates interleukin-12 and interleukin-10 production in leprosy.

The ability of monocytes to influence the nature of the T cell response to microbial pathogens is mediated in part by the release of cytokines. Of particular importance is the release of IL-12 and IL-10 by cells of the monocyte/macrophage lineage upon encountering the infectious agent. IL-12 promotes cell mediated immunity (CMI) to intracellular pathogens by augmenting T-helper type 1 responses, whereas IL-10 downregulates these responses. The ability of IFN-gamma to modulate the balance between IL-12 and IL-10 production was examined by studying leprosy as a model. In response to Mycobacterium leprae stimulation, IFN-gamma differentially regulated IL-12 and IL-10 production resulting in upregulation of IL-12 release and downregulation of IL-10 release. Furthermore, we determined that the mechanism by which IFN-gamma downregulates IL-10 was through the induction of IL-12. The data suggest a model of lymphocyte-monocyte interaction whereby the relative presence or absence of IFN-gamma in the local microenvironment is a key determinant of the type of monocyte cytokine response, and hence the degree of CMI in the host response to infection.

[The expression of ICAM-1 on macrophages stimulated with Mycobacterium avium complex and its control by some regulatory cytokines].

The interaction of LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells (APCs) is critical in determining conjugate formation between the APCs and T cells as well as activation of T cells. Recently, it was found that stimulation of THP-1 cells, a human monocyte M phi cell line, with Mycobacterium tuberculosis or its lipoarabinomannan, elicited the increase in the ICAM-1 expression. In addition, in cases of lepromatous leprosy patients with a serious defect in the M. leprae antigen-specific cellular immunity, keratinocytes in the leprosy lesions were lacking in the ICAM-1 expression. Therefore, ICAM-1 seems to participate in the host response to mycobacterial infections. Here, we studied the mode of the expression of ICAM-1 molecules on murine peritoneal M phis in response to stimulation with M. avium complex (MAC). In addition, the regulatory effect of some cytokines including TNF-alpha, IL-10, and transforming growth factor-beta (TGF-beta) on the ICAM-1 expression was studied. Monolayer cultures of peptone-starch induced murine peritoneal M phis were cultured in RPMI-1640 medium in the presence of MAC with or without test agents. At intervals, the M phis were stained with anti-ICAM-1 antibody (Ab) and then treated with alkaline phosphatase (Ap)-conjugated anti-1g Ab. After color development with NBT-BCIP substrate, percentage of the blue-stained (ICAM-1 positive) M phis was determined microscopically. The concentrations of TNF-alpha, IL-10, and TGF-beta in the M phi culture fluid was measured by ELISA using capture Ab, biotin-labelled capping Ab, and Ap-conjugated streptavidin. When M phis infected with MAC organisms were cultured in RPMI medium containing 10% fetal bovine serum or in serum-free GPI medium, a significant increase in their ICAM-1 expression was observed, reaching the peak at days 1 to 3, thereafter rapidly decreased and returned to the normal level by day 14. Further addition of TNF-alpha caused no significant change in the mode of the MAC-induced expression of ICAM-1. A transient increase in the IL-10 production of MAC-infected M phis was observed during the first 3-days cultivation, as in the case of TNF-alpha. On the other hand, TGF-beta production of the Mø population was initiated from day 3, and therafter increased gradually until day 14. ICAM-1 expression of the MAC-infected M phis was not influenced by the addition of IL-10, while anti-IL-10 Ab retarded the decline of ICAM-1 expression at day 7. On the other hand, the addition of TCF-beta attenuated the MAC infection-induced increase in the ICAM-1 expression significantly. In addition, anti-TGF-beta Ab significantly delayed the reduction of the ICAM-1 expression of MAC-infected M phis during days 3 to 7. These results indicate that, in the MAC-infected M phis. TGF-beta rather than IL-10 play important roles as a mediators for the late-phase down-regulation of ICAM-1 expression which had been transiently elevated by the action of TNF-alpha in the early phase of the Mø cultivation.