Increased chitotriosidase activity in serum of leprosy patients: Association with bacillary leprosy

Human phagocyte-specific chitotriosidase is associated with several diseases involving macrophage activation. Since macrophage activation plays an important role in the control of Mycobacterium leprae infection, we studied the association of chitotriosidase with leprosy both in serum and in situ in lesional skin biopsies from patients. Serum samples from 78 Indonesian leprosy patients (39 non-reactional and 39 reactional leprosy patients) and 36 healthy controls (HC) from the same endemic region were investigated. The patients were classified as multibacillary (MB, n = 69) or paucibacillary (PB, n = 9) based on the bacterial index in slit-skin smears. Thirty-six of the reactional patients had erythema nodosum leprosum (ENL), while only 3 had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 patients with ENL and one with RR. Multibacillary (MB) patients showed increased chitotriosidase activity in serum as compared to paucibacillary (PB) patients and healthy controls. Although no significant difference was observed between reactional and the corresponding non-reactional groups, ENL showed significantly higher chitotriosidase activity as compared to HC. Furthermore, corticosteroid treatment resulted in significant decline of enzyme activity in ENL sera. Chitotriosidase activity correlated with levels of neopterin, another macrophage activation marker, but not with IL-6, IFN-γ, TNF-α and IL-10. Immunohistochemical staining of 6 MB (LL = 5, BL = 1) lesional skin sections from stored material showed positive staining for chitotriosidase within lipid-laden macrophages suggesting that macrophages are the source of the enzyme detected in serum. Thus, serum chitotriosidase activity is potentially useful in distinguishing MB from PB leprosy and in monitoring response to therapy in ENL.     

Cytokine mRNA expression in leprosy: a possible role for interferon-gamma and interleukin-12 in reactions (RR and ENL)

Leprosy patients during the natural course of the disease may develop reactional episodes, namely reversal reaction (RR) and erythema nodosum leprosum (ENL). Immunological events described as occurring during RR indicate up-regulation of the immune response, whereas in ENL the events are not fully understood. The aim of this study was to analyse the in vivo pattern of cytokine gene expression in the reactional states of leprosy. Peripheral blood mononuclear cells (PBMC, n = 14) and tissue samples (n = 17) obtained from patients with ENL and RR were obtained and assayed by RT-PCR. PBMC obtained from unreactional patients (n = 15) and normal individuals (n = 5) were also assessed. Expression of interferon (IFN)gamma, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-2Rp55, perforin and IL-1beta mRNA in PBMC were detected mostly in ENL/RR patients, but not in unreactional patients. Likewise, cytokines such as IL-6, IL-8, tumour necrosis factor (TNF)alpha and TNFbeta were also present in reactional and tuberculoid patients as opposed to lepromatous leprosy (BL/LL). Interestingly, the majority of ENL/RR patients showed messages for IL-6, IL-10, IL-12 and TNFalpha in the skin. IFNgamma was detected in 84.6% (ENL) and 100% (RR) of the patients, whereas IL-4 was detected only in few individuals (38.5 and 25%, respectively). Although mRNA expression and protein levels may be different, the data reported in this study suggest a cytokine mRNA profile that seems to be indistinguishable for RR and ENL. In addition, it shows up-regulation of immuno-inflammatory cytokines in the blood and tissue of the same patient examined before and during reaction. Furthermore, it is suggested that this pattern of response results from an immunological reactivation that might lead to an acute inflammatory response in both reactional episodes.     

Concentrations of cytokines,soluble interleukin-2 receptor,and soluble CD30 in sera of patients with hepatitis B virus infection during acute and convalescent phases

The immunoregulatory roles of interleukin-2 (IL-2), IL-4, IL-10, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), the soluble form of the IL-2 receptor (sIL-2R), and the soluble form of CD30 (sCD30) were evaluated in patients with hepatitis B virus (HBV) infection. Two groups of subjects were studied: 15 healthy individuals without hepatitis antecedents and 15 patients with HBV infection. Blood samples were taken during the acute and convalescent phases. The analysis of the samples was done by the enzyme-linked immunosorbent assay technique. IFN-gamma and TNF-alpha levels decreased in the convalescent phase. IL-10, IL-2, and sIL-2R levels increased in the acute and convalescent phases, while sCD30 levels increased during the acute phase. The IL-4 concentrations decreased in both phases. During the acute phase, IFN-gamma and TNF-alpha induced increases in IL-2, sIL-2R, IL-10, and sCD30 levels in serum, which allowed the development of immunity characterized by the nonreactivity of the HBV surface antigen, the onset of antibodies to the HBV surface antigen (anti-HBs), and normal alanine aminotransferase levels during the convalescent phase. Increased IL-2 levels during the acute phase would stimulate the activities of NK cells and CD8(+) lymphocytes, which are responsible for viral clearing. The raised sIL-2R levels reveal activation of T lymphocytes and control of the IL-2-dependent immune response. The sCD30 increment during the acute phase reflects the greater activation of the Th2 cellular phenotype. Its decrease in the convalescent phase points out the decrease in the level of HBV replication. The increase in IL-10 levels could result in a decrease in IL-4 levels and modulate IFN-gamma and TNF-alpha levels during both phases of disease, allowing the maintenance of anti-HBs concentrations.     

Effect of thalidomide on the expression of TNF-alpha m-RNA and synthesis of TNF-alpha in cells from leprosy patients with reversal reaction

Hypersensitivity reactions called reversal reaction (RR) and erythema nodosum leprosum (ENL) occur in leprosy. They are characterized by an increase in tumor necrosis factor-alpha (TNF-alpha). Thalidomide is an effective treatment for ENL but not RR. Its effectiveness in ENL is attributed to inhibition of TNF-alpha, and this does not explain its failure to treat RR. We assessed thalidomide's effect on TNF-alpha in RR. Mononuclear cells from RR and non-RR patients and healthy individuals were treated with thalidomide and M.leprae (AFB), a cytosol fraction of M. leprae or Dharmendra lepromin. Thalidomide suppressed TNF-alpha, but when some RR patients' cells were stimulated with AFB, it enhanced TNF-alpha.     

Interleukin 6, tumor necrosis factor alpha and their soluble receptors in the blood serum of patients with denture stomatitis and fungal infection

Determinations of the blood serum levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and their soluble receptors (sIL-6R, sTNFR) in denture stomatitis patients (DS) were performed. Serum levels of interleukins and their soluble receptors were measured using the ELISA method. In all examined patients mycological diagnostics were conducted using API 20C AUX stripe tests and an automatic ATB machine. Results were compared with those of healthy denture wearers (D), and controls (C). In DS patients, yeasts were isolated in 90.9%, in D in 66.7% of cases. The most often isolated species in both groups was Candida albicans. Mean concentrations of IL-6 and TNF-alpha were statistically significantly higher in DS and D groups compared to controls. Mean concentrations of sIL-6R were similar in all groups; however, concentrations of sTNFR in both DS and D groups were significantly lower compared to controls. There were no correlations found between values of IL-6 and TNF-alpha nor between examined interleukins and their soluble receptors.     

High levels of inflammatory cytokines are associated with poor clinical response to steroid treatment and recurrent episodes of type 1 reactions in leprosy

Levels of leprosy antigen-induced interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) were measured in 96 leprosy patients with type 1 reactions (T1R) before, during and after a standard 12-week course of steroids. Peripheral blood mononuclear cells (PBMC) from leprosy patients with untreated T1R produced significantly more TNF-alpha than leprosy patients without T1R. Median levels of IFN-gamma and TNF-alpha in T1R patients fell during treatment with steroids; however, TNF-alpha levels increased as the steroid dose was reduced. Median IL-10 levels increased throughout the steroid treatment period and were associated strongly with TNF-alpha levels. Patients with high cytokine levels had a poorer recovery of sensory or voluntary muscle nerve function, a higher risk of reactivation of symptoms during steroid treatment, and a higher risk of another episode of T1R within 2 months of completing the steroid regimen. Rapid and effective reversal of the inflammatory process in T1R is critical to prevent permanent nerve damage from T1R and monitoring cytokine levels during treatment may be useful.     

Patterns of cytokines and soluble cellular receptors in the sera of children with acute chagas' disease

Cytokines and soluble cellular receptors are involved in inflammatory processes and probably in the pathogenesis of parasite and bacterial diseases. In a previous study, we reported increased levels of soluble receptors of interleukin-2 (sIL2-R) in children with acute Chagas' disease, one of the main parasitic infections that is endemic in Latin America. We sought to analyze the pattern of different cytokines and soluble receptors in the sera of children with chagasic infection. Children with acute and indeterminate stages of Chagas' disease, as well as nonchagasic children, were studied. Sera were assayed by enzyme-linked immunosorbent assay to measure the levels of tumor necrosis factor alpha (TNF-alpha), IL-6, IL-2, IL-8, IL-12, sIL-2R, and the soluble receptors of CD8 and CD4 (sCD8 and sCD4). sIL-2R and sCD8 showed the highest levels in serum in acutely infected children, decreasing after specific antiparasite therapy. Chronic children showed a pattern similar to the one of nonchagasic children. Although they were not statistically significant, TNF-alpha, IL-6, and sCD4 showed a tendency to reach high levels in the acutely infected group, whereas IL-2, IL-8, and IL-12 did not reveal changes with respect to the noninfected children. In summary, we report here the patterns of cytokines and soluble receptors in in the sera of children infected with Trypanosoma cruzi; we found significantly increased levels of sIL-2R and sCD8 in acute infection that decreased after therapy, and high levels of TNF-alpha, IL-6, and sCD4 in some of the acute patients. The measurement of sIL-2R and sCD8 may provide a useful tool in the follow-up of children with Chagas' disease.     

Different lymphoid cell populations produce varied levels of neopterin, beta 2-microglobulin and soluble IL-2 receptor when stimulated with IL-2, interferon-gamma or tumour necrosis factor-alpha.

Immune activation is central to many immune disorders. Clinical investigations have shown that immune activation can be quantified by measurements of soluble immune activation products in serum. Most in vitro studies of these immune activation products have focused on single products. In this study the specific cell sources and the major lymphokines inducing multiple activation products were investigated. In vitro addition of interferon-gamma (IFN-gamma) or IL-2 stimulated peripheral blood mononuclear cells to produce neopterin, beta 2-microglobulin (beta 2-M) and soluble IL-2 receptor (sIL-2R). These two lymphokines can act independently, because neutralizing antibodies to one of the lymphokines did not block the inducing activity of the other. Tumour necrosis factor-alpha (TNF-alpha) was also investigated and shown to be a less powerful inducer than IL-2 or INF-gamma. Separated lymphoid subpopulations responded differently to specific lymphokines. Monocytes produced only neopterin and only in response to INF-gamma. T cells released beta 2-M and sIL-2R in response to IL-2. B cells, however, were capable of producing all three immune activation products. Neopterin production in B cells was induced by either INF-gamma of IL-2, indicating that B cells have additional mechanisms for responding to lymphokines. To investigate whether these in vitro findings also occur in vivo, sera from patients who had received either rIL-2 or INF-gamma treatment were tested. INF-gamma administration led to substantial increases in serum neopterin but only a moderate beta 2-M increase and no increase in the serum sIL-2R levels. rIL-2 administration caused a substantial increase of all three serum immune activation products, consistent with our in vitro findings. The results confirm that increased serum levels of soluble immune activation products are indicators of increased cytokine production by lymphocytes and monocytes and also that B cells can be a prominent source of immune activation products.     

The study of immunological markers in patients with "intrinsic" type atopic dermatitis]

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by several clinical, immunological and biochemical alterations. Comparing the patients with the 'extrinsic' and 'intrinsic' types of AD, we investigated the role of immunological mechanisms in the pathogenesis of AD. To confirm it, we calculated serum markers of T lymphocyte activation: soluble interleukin-2 receptor (sIL-2R), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-gamma (IFN-gamma). The soluble CD14 (sCD14) and tumor necrosis factor-alpha (TNF-alpha) in serum were measured as monocyte/macrophage activation markers. We examined 29 patients with the 'extrinsic' type AD (serum IgE > 10000 IU/ml: High-AD), 23 patients with the 'intrinsic' type AD (serum IgE < 37 IU/ml: Low-AD) and 11 healthy controls. Serum sIL-2R levels were increased in High-AD and Low-AD compared with the controls. They were also significantly increased in High-AD compared with Low-AD. Serum sCD14 levels were increased in High-AD compared with Low-AD and the controls. Severity index of AD were correlated with serum sIL-2R levels but not with sCD14 levels in sera. In conclusion, IgE may not relate with the pathogenesis of atopic dermatitis. Serum sIL-2R levels may be increased according to inflammatory skin lesions and it may be exaggerated with the immunological activation in the patients with the 'extrinsic' type AD.     

The significance of serum soluble IL-2 receptor as a marker for active visceral leishmaniasis in Sicilian patients.

Sera from nine Sicilian patients with confirmed visceral leishmaniasis (Leishmania donovani infantum; VL), at the moment of the diagnosis, during the course of the disease and after clinical recovery, were analysed for the concentration of soluble IL-2 receptor (sIL-2R). The results show that sIL-2R is a marker of disease activity, since it is in high concentration at the beginning of infection and returns to the normal range following successful chemotherapy. At the same time of serum analysis for sIL-2R, peripheral blood mononuclear cells (PBMC) of VL patients were stimulated with phytohaemagglutinin (PHA) or antigen and supernatant tested for IL-2 and interferon-gamma (IFN-gamma) production. Data demonstrate that there is an inverse relation between concentration of IL-2 and IFN-gamma in the supernatants and sIL-2R secretion in the sera.     

IL-6 inhibits the proliferation of fibroblastic synovial cells from rheumatoid arthritis patients in the presence of soluble IL-6 receptor

IL-6 and tumor necrosis factor (TNF)-alpha have been proven to play an important role in the development of rheumatoid arthritis (RA). It is well known that TNF-alpha induces IL-6 production from synovial cells as well as their proliferation. The effect of IL-6 on synovial cells, however, is not clear. An in vitrostudy was performed to determine the effect of IL-6 on the proliferation of synovial cells. Fibroblastic synovial cells isolated from the synovial tissues of eight RA patients were employed after the third to sixth passages. IL-6 in the presence of soluble IL-6 receptor (sIL-6R) inhibited the proliferation of synovial cells in a dose-dependent manner in seven cases without increasing the number of necrotic or apoptotic cells, while TNF-alpha increased synovial cell proliferation in all cases. The inhibitory effect of IL-6 was observed only in the presence of sIL-6R although small amounts of IL-6R were detected in these cells by RT-PCR analysis. However, anti-IL-6R or anti-gp130 mAb treatment increased spontaneous growth of synovial cells in all eight cases, suggesting that endogenous IL-6 and a small amount of IL-6R expressed in synovial cells suppressed their growth without exogenous IL-6 or sIL-6R. In addition, the IL-6-sIL-6R complex reduced the TNF-alpha-induced proliferation of synovial cells while TNF-alpha induced their IL-6 production. These data suggest that IL-6 may act as a negative feedback factor for TNF-alpha-induced synovial cell growth.     

Sera of leprosy patients with type 2 reactions recognize selective sequences in Mycobacterium leprae recombinant LSR protein.

Type 2 reactions (erythema nodosum leprosum [ENL]) are episodic, reactional states causing significant morbidity in lepromatous leprosy patients. With a view to defining the immunological differences between the stable and reactional forms of lepromatous leprosy, we determined antibody responses to LSR, a recombinant protein of Mycobacterium leprae previously described by us (S. Laal, Y.D. Sharma, H.K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. S. Mishra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991), as well as to 10- to 15-mer overlapping peptides synthesized on the basis of the LSR amino acid sequence. We report here the selective recognition of B cell epitopes by sera from patients with ENL as compared with a control group with nonreactional lepromatous leprosy. Peptides 2 and 3, with the sequences GVTYEIDLTNKNAA and IDLTNKNAAKLRGD, respectively, were recognized by > 95% of sera from patients with active ENL. Peptide 3 in addition showed reactivity with sera taken from 91.6% of lepromatous leprosy patients who were apparently stable but who were recorded to have had ENL several weeks before or after the sample collection. The core sequence IDLTNKNAA common to both these peptides may be a major target of humoral responses in ENL. In addition, the RGD motif at the C terminus appeared to influence the antigenicity of peptide 3 in enzyme-linked immunosorbent assay. It would appear that humoral responses during ENL are directed to selective antigenic determinants of the leprosy bacillus. The use of such serological markers to identify lepromatous leprosy patients with a high risk for developing ENL would be of clinical and predictive value.     

TNF-alpha, IL-6 and their soluble receptor serum levels and secretion by neutrophils in cancer patients

Simultaneous evaluation of cytokines and their soluble receptor production and the serum levels can be helpful in understanding the local and systemic immune response of a tumor-bearing host. In the present study we examined serum levels of TNF-alpha, IL-6 and their soluble receptors: sTNFRp55, sTNFRp75 and sIL-6R confronted with their production by the polymorphonuclear neutrophils (PMN) from cancer patients. Examinations were carried out in patients with adenocarcinoma breast cancer and squamous cell carcinoma of the oral cavity and related to the clinical course and to different phases of therapy. Secretion of IL-6, sTNFRp55 and sTNFRp75 by PMN appeared to be dependent on tumor type, clinical progression of disease as well as on therapy, suggesting a significant role of these cells at different phases of the immune response to cancer associated with these mediators. Changes in values of TNF-alpha, IL-6 and their soluble receptors in sera of both cancer groups, dependent on tumor type, clinical progression and cancer therapy, could have a diagnostic and prognostic role in cancer disease.     

Neutrophils isolated from leprosy patients release TNF-alpha and exhibit accelerated apoptosis in vitro

This study demonstrated that polymorphonuclear neutrophils (PMN) participate in the acute inflammatory response in leprosy as effector cells. Lepromatous patients present intense infiltrate of neutrophils in reactional (ENL) lesions. Circulating PMN of nonreactional patients, healthy donors, and reactional patients were purified and analyzed in vitro. The study confirmed the short lifespan of these cells in culture with progressive changes characteristic of apoptosis. Apoptosis was greatly accelerated in ENL patients as shown by cellular morphology, later confirmed by qualitative and quantitative analysis of fragmented DNA. It was observed that neutrophils stimulated with lipopolysaccharide, Mycobacterium leprae, and lipoarabinomannan secrete interleukin-8 and tumor necrosis factor alpha (TNF-alpha). Thalidomide, a drug known to inhibit TNF-alpha synthesis on monocytes, also exerted an inhibitory effect on TNF-alpha secretion in neutrophils. These data suggest that PMN can participate in the regulation of the immune response in leprosy and can contribute to the amplification of TNF-alpha production at the site of ENL lesion.     

Some immunological aspects in the reaccional states of Hansen disease

The crucial clinical problem in leprosy is represented by episodes of intense inflammation that produce nerve damage. When Mycobacterium leprae has been eliminated by means of antibiotics, the death of the bacteria is not a complete solution to the damage caused in nerves. Two of the more frequent immunopathological phenomena in Hansen's disease are Type I, reactions, known as Reversal Reactions (RR), and Type II reactions, of which the most frequent and well known are those called Erythema Nodosum Leprosum (ENL). Type II reactions have been defined as an immunologic complication in multibacillary patients. Both types of reactions are accompanied mainly by the increase of pro-inflammatory cytokines TNF-alpha, IL-1, IL-2, IL-4, IL-6, IL-8, INF-gamma, IL-10, IL-12, among others. In a retrospective analysis of a population of 150 patients of the Central Service of Dermatology, Institute of Biomedicine, where these patients were subjected to Multidrug Therapy (MDT) and MDT + inmunotherapy (IMT), both groups presented type II reactions, but the group that additionally received inmunotherapy also presented type I reactions. In the group of patients that presented ENL, it appeared to be associated with the large amount of bacilli in their lesions.     

Serum levels of tumour necrosis factor-alpha and interleukin-1 beta during leprosy reactional states.

The possible role of cytokines in leprosy reactions was investigated by analysing the levels of tumour necrosis factor (TNF) and interleukin-1 (IL-1) in serum samples from 39 leprosy patients, 22 of them presenting either type I (upgrading) or type II (ENL) reactions. Fifty per cent of the patients showed elevated concentrations of TNF and IL-1 in at least one of the serum samples tested. This included all four patients undergoing type I reversal reaction and nine (50%) of the ENL patients studied. Concentrations of TNF above 1000 pg/ml were found in four patients with ENL. Development of erythema multiforme in these ENL patients represented an aggravating factor and all four patients suffering from this type of lesion demonstrated increased serum TNF levels. All BT patients tested presented elevated IL-1 levels, while only half of them presented elevated levels of TNF. No correlation was found between any particular systemic symptoms and the levels of TNF and IL-1. These results suggest that TNF and IL-1 may be implicated in leprosy reactions, either acting directly or in synergism with other cytokines.     

Role of neutrophils in release of some cytokines and their soluble receptors

Available data suggest that cytokines and soluble cytokine receptors, which are being produced by immunological cells, can modulate the immune response of the host. Although the production of mediators such as TNF-alpha and IL-6 as well as that of their soluble receptors has been extensively studied in tissue cells and mononuclear cells, it has not been fully investigated in neutrophils (PMN). In the present study we examined the ability of PMN to simultaneously release TNF-alpha, IL-6 and their soluble receptors-sTNFRp55, sTNFRp75 and sIL-6R. Concentrations of soluble receptors were compared with expression of membrane-bound TNF and IL-6 receptors. For comparative purposes, similar examinations with autologous peripheral blood mononuclear cells (PBMC) were performed. We found that PMN and PBMC have the same ability to release IL-6 and sTNFRp75. In contrast, there were significant differences in the release of TNF-alpha, sTNFRp55 and sIL-6R between these cells. Reduction in membrane TNF receptor expression, observed in this study, was associated with increase secretion of soluble TNF receptors by PMN and PBMC. The results suggest that PMN can play an essential role in modulating the inflammatory response by affecting the balance between pro-inflammatory mediators, such as TNF-alpha, and anti-inflammatory mediators, such as soluble TNF receptors.     

Protein profile in leprosy

Serum proteins and plasma fibrinogen were estimated in 103 patients in various groups of leprosy and 52 patients of reactional leprosy. Total proteins, serum globulin and fibrinogen showed significant rise while serum albumin showed fall over the immunological spectrum from TT to LL. Type II reactional leprosy similarly revealed significant rise in globulin and fibrinogen. The comparison of these parameters between most of the comparable groups of leprosy was statistically significant. ENL patients after complete subsidence of reaction and after steroid treatment showed significant decrease in these protein fractions, thus conferring some prognostic implication on these tests.     

Natural emergence of antigen-reactive T cells in lepromatous leprosy patients during erythema nodosum leprosum.

Fifteen lepromatous leprosy (LL) patients undergoing erythema nodosum leprosum (ENL) reactions were compared with 13 stable, uncomplicated, anergic individuals of the same leprosy background. ENL patients showed significant antigen-induced leukocyte migration inhibition (migration index = 0.058 +/- 0.01), paralleling the values obtained with a responder tuberculoid leprosy population (migration index = 0.04 +/- 0.004). Both phytohemagglutinin-induced general T-cell proliferation and, more significantly, antigen-induced lymphoproliferation were enhanced during the acute phase of the reaction. Suppressor cell activity, monitored by a costimulant assay, showed enhanced antigen-stimulated suppression of mitogen responses. Interestingly, the improvement in in vitro T-cell responses was not reflected in dermal reactivity, since 48-h delayed-type hypersensitivity responses after intradermal injection of soluble Mycobacterium leprae antigens continued to be poor. After subsidence of reactional lesions, leukocyte migration inhibition, lymphoproliferation, and suppressor cell activity were reduced to the unresponsive state seen in stable LL patients. Significantly, perturbations of T-cell reactivity are detectable in ENL reactions, indicating the natural but transient emergence of antigen-induced T cells in LL.