Erythema Nodosum Leprosum Lesions Research Articles

Image Morphometric Analysis of B Cells and Plasma Cells in Erythema Nodosum Leprosum With Clinicopathological Correlation.

Erythema nodosum leprosum (ENL) occurs as an immunological complication of multibacillary leprosy (MBL). The pathogenesis of ENL is long considered to be a T-cell-mediated process. The role of B cells and plasma cells in ENL is not well described in the literature. Therefore, we investigated the B-cell and plasma cell infiltrates in the skin biopsies of biopsy-proven cases of ENL by immunohistochemistry and image morphometry and compared the result with paucibacillary leprosy and MBL. Moreover, we sought a correlation of the B-cell and plasma cell infiltrates with different clinical, hematological, histopathological, and bacteriological parameters as well as the T-cell subsets in the skin biopsies. Our study highlighted a significant reduction in the number of B cells from paucibacillary leprosy to MBL to ENL, although there was no significant variation in the plasma cell infiltrate. The plasma cell infiltrate correlated with absolute neutrophilia in the blood and the presence of eosinophils in the ENL lesions. Both B cells and plasma cells positively correlated with CD4-positive T-helper cells and the CD8-positive cytotoxic T cells. Besides, the B cells also correlated positively with the CD3-positive pan T cells in the biopsy and negatively correlated with the T-regulatory:T-cell ratio. Our results suggested the role of B cells and plasma cells even at the tissue level in the pathobiogenesis of ENL.

Elevated Pentraxin-3 Concentrations in Patients With Leprosy: Potential Biomarker of Erythema Nodosum Leprosum.

Leprosy, the leading infectious cause of disability worldwide, remains a major public health challenge in the most severely affected countries despite the sharp decline in new cases in recent years. The search for biomarkers is essential to achieve a better understanding of the molecular and cellular mechanisms underlying the disease. Pentraxin-3 (PTX3) analyses of sera from 87 leprosy patients with or without reactions were conducted via enzyme-linked immunosorbent assay. In situ identification of PTX3 in skin lesion was confirmed by quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunofluorescence assays. We found that PTX3 serum levels were higher in multibacillary patients when evaluated before the onset of acute erythema nodosum leprosum (ENL) and persistently elevated during reaction. Thalidomide treatment reduced PTX3 in the serum 7 days after starting treatment. In situ analyses have also demonstrated enhancement of PTX3 in ENL lesions and showed that treatment with thalidomide reduced its expression and the prominent neutrophilic infiltrate, a hallmark of the disease. In summary, our study provides in vivo evidence that PTX3 is enhanced during ENL but not in reversal reaction and provides a new molecular target in ENL pathogenesis.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Erythema nodosum leprosum limited to sun-exposed sites: An unusual presentation of type 2 lepra reaction

The distribution of erythema nodosum leprosum lesions limited to sun-exposed sites is an unusual presentation of a type 2 reaction. We report the case of a 23-year-old female who presented with erythematous papules, nodules and plaques confined to sun-exposed areas over the face, extensor aspect of both arms and forearms, and “V” of chest with fever and joint pains as well as ulnar clawing of the left hand. A diagnosis of type 2 lepra reaction was made which was confirmed by slit skin smear and skin biopsy.

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably contributing to the immunopathogenesis of the disease.

Integrated Pathways for Neutrophil Recruitment and Inflammation in Leprosy

Neutrophil recruitment is pivotal to the host defense against microbial infection, but it also contributes to the immunopathology of disease. We investigated the mechanism of neutrophil recruitment in human infectious disease by means of bioinformatic pathways analysis of the gene expression profiles in the skin lesions of leprosy. In erythema nodosum leprosum (ENL), which occurs in patients with lepromatous leprosy and is characterized by neutrophil infiltration in lesions, the most overrepresented biological functional group was cell movement, including E-selectin, which was coordinately regulated with interleukin 1beta (IL-1beta). In vitro activation of Toll-like receptor 2 (TLR2), up-regulated in ENL lesions, triggered induction of IL-1beta, which together with interferon gamma induced E-selectin expression on and neutrophil adhesion to endothelial cells. Thalidomide, an effective treatment for ENL, inhibited this neutrophil recruitment pathway. The gene expression profile of ENL lesions comprised an integrated pathway of TLR2 and Fc receptor activation, neutrophil migration, and inflammation, providing insight into mechanisms of neutrophil recruitment in human infectious disease.

Towards understanding the pathology of erythema nodosum leprosum

Erythema nodosum leprosum (ENL) is an immune-mediated complication of leprosy presenting with inflammatory skin nodules and involvement of multiple organ systems, often running a protracted course. Immune complex production and deposition as well as complement activation have long been regarded as the principal aetiology of ENL. However, new data show that cell-mediated immunity is also important. We have performed a critical analysis of studies on the pathology of ENL. Our main findings are as follows. ENL is characterised by an inflammatory infiltrate of neutrophils with vasculitis and/or panniculitis. There is deposition of immune complexes and complement together with Mycobacterium leprae antigens in the skin. Changes in serum levels of Igs indicate a transient, localised immune response. The major T-cell subtype in ENL is the CD4 cell, in contrast to lepromatous leprosy where CD8 cells predominate. The cytokines TNFalpha and IL-6 are consistently found whilst IL-4 is low or absent in ENL lesions, indicating a T(H)1 type response. Keratinocyte 1a and intercellular adhesion molecule-1 (ICAM-1) have been shown to be present in the epidermis in ENL, which is evidence of a cell-mediated immune response. Co-stimulatory molecules such as B7-1 have also been studied but further work is needed to draw strong conclusions. We also highlight potential areas for future research.

E‐selectin induction by IL‐1 and IFN‐ γ in the pathogenesis of type III immune hypersensitivity

Erythema nodosum leprosum (ENL), a reaction in lepromatous leprosy (L‐lep), is a prototypical type III hypersensitivity reaction characterized by immune‐complex deposition and a neutrophilic infiltrate. We investigated the differential gene expression profiles of L‐lep and ENL lesions; both supervised and unsupervised analyses indicated that the different patient groups express a distinct lesional gene profile. The cell adhesion gene cluster, including e‐selectin (E‐sel), was the most overrepresented biological process increased in ENL compared to L‐lep. E‐sel protein was expressed on blood vessels, and was comparatively higher in ENL than L‐lep lesions. IL‐1 and IFN‐γ induced high levels of E‐sel expression on endothelial cells and induced neutrophil adhesion. Thalidomide, an effective treatment for ENL, diminished IL‐1 and IFN‐γ induced E‐sel expression on endothelial cells and diminished neutrophil binding. These data suggest a role for IL‐1 plus IFN‐γ induction of E‐sel on endothelial cells in mediating neutrophil recruitment in the pathogenesis of ENL and type III hypersensitivity reactions.

Role of azathioprine in preventing recurrences in a patient of recurrent erythema nodosum leprosum

The pathogenesis of erythema nodosum leprosum (ENL) involves both immune complex deposition and dysfunction of cell mediated immunity. Tumour necrosis factor-alpha (TNF-alpha) plays an important role in its pathogenesis. Thalidomide and corticosteroids are the mainstay of treatment for ENL. However, there are often severe limitations to their use. We report a case of recurrent ENL treated successfully with azathioprine. A 15-year-old unmarried girl with lepromatous leprosy had recurrent ENL for 2 years. She was treated with WHO-MB MDT and prednisolone in doses of 40-90 mg a day for 2-12 weeks. Her condition was inadequately controlled. The patient was therefore treated with thalidomide 300 mg and prednisolone 40 mg. The symptoms subsided after 5 days and ENL lesions healed in 2 weeks. Prednisolone was reduced by 10 mg per week and stopped, while thalidomide was reduced to 100 twice daily after 4 weeks. Azathioprine 100 mg (2 mg/kg per day) daily orally was added to prevent recurrences. Thalidomide was further reduced and stopped after another 4 weeks while she continued with azathioprine in the same doses for 8 months. There was no recurrence of ENL lesions and no side effects of the therapy. MB-MDT was stopped 1 year ago, and she is on follow-up without any relapse. Azathioprine, therefore, appears to be an effective and safe drug to prevent recurrences of ENL.

Bullous erythema nodosum leprosum (bullous type 2 reaction)

A 35-year-old man suffering from lepromatous leprosy with recurrent erythema nodosum leprosum (ENL) (bullous type 2 reaction) was admitted for acute exacerbation of the reaction. He had received regular WHO multibacillary multidrug therapy (WHO-MB-MDT) and corticosteroids for 11 months. The dose of corticosteroids could not be tapered below 20 mg/day due to sudden, frequent, and severe relapses of ENL. In hospital, he suddenly developed hypotension for which he was moved to the intensive care unit and managed. On the third day, he developed multiple, flaccid and tense bullae, some of which were hemorrhagic, over the face, trunk, and proximal extremities (Fig. 1). Some of the ENL lesions showed a central bluish hue. Investigations revealed leukocytosis, thrombocytopenia, and hypokalemia. Blood culture yielded coagulase-negative Staphylococcus aureus. Slit skin smear and smear from the blister fluid showed numerous, fragmented, acid-fast bacilli (AFB) in clusters and many neutrophils (Fig. 2). Skin biopsy revealed a subepidermal bulla that consisted of neutrophils and macrophage granulomas in the dermis together with perivascular neutrophilic infiltration (Fig. 3); however, there were no features suggestive of vasculitis. Fite's stain demonstrated fragmented AFB. Both direct and indirect immunofluorescence studies did not show features of autoimmune bullous disorders; however, moderately strong discontinuous fibrinogen deposits were seen along the basement membrane and around the blood vessels. There were no deposits of immunoglobulin G (IgG) and C3. He was treated with antimicrobials, potassium chloride, and hydrocortisone, 100 mg intravenously, twice daily, which was subsequently switched to oral prednisolone. He responded well and was discharged. Upon lowering the dose of corticosteroids, he again developed severe ENL with fewer bullous lesions. Figure Figure 1 Open in figure viewerPowerPoint Multiple, tense, hemorrhagic bullae over the trunk Figure 2Open in figure viewerPowerPoint Smear from the blister fluid showing numerous, fragmented, acid-fast bacilli (AFB) and neutrophils (Ziehl–Neelsen stain, × 200) Figure 3Open in figure viewerPowerPoint Photomicrograph of the skin biopsy showing subepidermal bulla consisting of neutrophils and macrophage granulomas in the dermis together with perivascular neutrophilic infiltration (hematoxylin and eosin, × 200)

Bullous erythema nodosum leprosum (bullous type 2 reaction)

A 35‐year‐old man suffering from lepromatous leprosy with recurrent erythema nodosum leprosum (ENL) (bullous type 2 reaction) was admitted for acute exacerbation of the reaction. He had received regular WHO multibacillary multidrug therapy (WHO‐MB‐MDT) and corticosteroids for 11 months. The dose of corticosteroids could not be tapered below 20 mg/day due to sudden, frequent, and severe relapses of ENL. In hospital, he suddenly developed hypotension for which he was moved to the intensive care unit and managed. On the third day, he developed multiple, flaccid and tense bullae, some of which were hemorrhagic, over the face, trunk, and proximal extremities (Fig. 1). Some of the ENL lesions showed a central bluish hue. Investigations revealed leukocytosis, thrombocytopenia, and hypokalemia. Blood culture yielded coagulase‐negative Staphylococcus aureus. Slit skin smear and smear from the blister fluid showed numerous, fragmented, acid‐fast bacilli (AFB) in clusters and many neutrophils (Fig. 2). Skin biopsy revealed a subepidermal bulla that consisted of neutrophils and macrophage granulomas in the dermis together with perivascular neutrophilic infiltration (Fig. 3); however, there were no features suggestive of vasculitis. Fite's stain demonstrated fragmented AFB. Both direct and indirect immunofluorescence studies did not show features of autoimmune bullous disorders; however, moderately strong discontinuous fibrinogen deposits were seen along the basement membrane and around the blood vessels. There were no deposits of immunoglobulin G (IgG) and C3. He was treated with antimicrobials, potassium chloride, and hydrocortisone, 100 mg intravenously, twice daily, which was subsequently switched to oral prednisolone. He responded well and was discharged. Upon lowering the dose of corticosteroids, he again developed severe ENL with fewer bullous lesions.Multiple, tense, hemorrhagic bullae over the trunkimageSmear from the blister fluid showing numerous, fragmented, acid‐fast bacilli (AFB) and neutrophils (Ziehl–Neelsen stain, × 200)imagePhotomicrograph of the skin biopsy showing subepidermal bulla consisting of neutrophils and macrophage granulomas in the dermis together with perivascular neutrophilic infiltration (hematoxylin and eosin, × 200)image

Cytokine patterns of immunologically mediated tissue damage.

Reactional states in leprosy are produced by different immunologic mechanisms and are responsible for a major component of tissue damage of the disease. Reversal reactions exhibit increased CD4 T cell infiltration in lesions and augmented cell-mediated immune reactivity to Ag of Mycobacterium leprae that can rapidly produce nerve damage. Erythema nodosum leprosum (ENL) reactions also have CD4 T cell infiltration but appear to be associated with the formation of immune complexes that are responsible for panniculitis, arthritis, vasculitis, and nerve injury. Because these reactional states may serve as paradigms for other types of human immunologically mediated tissue damage, this study sought to characterize the dynamic changes in cytokines associated with these reactions. Expression of cytokine mRNA in lesions of leprosy reactional states were measured by PCR. In reversal reactions, IL-1 beta, TNF-alpha, IL-2, and IFN-gamma mRNA were prominent and found to increase during the reaction, concomitant with decreases in expression of mRNA for IL-4, IL-5, and IL-10. In ENL, selective increases in the expression of IL-6, IL-8, and IL-10 mRNA was observed, with persistent expression of IL-4 and IL-5 mRNA. Reversal reactions represent naturally occurring delayed-type hypersensitivity reactions that favor macrophage activation and protective immunity, but which can engender concomitant cell injury. In contrast, ENL lesions represent immediate-type hypersensitivity reactions reflecting the selective stimulation of cytokines that attract neutrophils, stimulate antibody production, and down-regulate macrophage activation. The analysis of cytokine dynamics within different inflammatory responses can provide insights into immune mechanisms of tissue damage, and provide a useful framework for developing strategies for therapeutic intervention.

Immune complexes and antibody levels in blisters over human leprosy skin lesions with or without erythema nodosum leprosum

Erythema nodosum leprosum (ENL) is a serous complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti- Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

Analysis of naturally occurring delayed-type hypersensitivity reactions in leprosy by in situ hybridization.

Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.

In situ and in vitro characterization of the cellular immune response in erythema nodosum leprosum.

We sought to evaluate cell-mediated immune responses in erythema nodosum leprosum (ENL), a reactional state occurring in lepromatous leprosy. Skin biopsies from patients with leprosy were studied with monoclonal antibodies against T lymphocyte antigenic determinants, interleukin 2 (IL 2), and IL 2 receptors (Tac) by using immunoperoxidase staining of frozen sections. Peripheral blood lymphocytes from 18 ENL patients were tested in vitro for lepromin-induced suppression of Con A stimulation. Serial studies of seven lepromatous patients who developed ENL during the course of the study showed increases in both the Leu-3a:Leu-2a ratio and the number of IL 2-positive cells. IL 2-positive cells comprised 0.3% of the cells in all of the ENL lesions studied as compared with the 0.03% found in nonreactional lepromatous lesions (P less than 0.001). Lepromin-induced suppression of the Con A response, present in nonreactional lepromatous patients, significantly decreased in patients developing the ENL reaction, but returned after recovery from ENL. These changes in tissues and peripheral blood suggest that the pathogenesis of ENL is related to cell-mediated immune processes. Despite these immunologic changes, however, ENL patients do not recover antigen-specific skin tests or eliminate Mycobacterium leprae.

Immunoglobulin deposits in Erythema Nodosum Leprosum (ENL) JOSEPHINE ANTHON

Erythema Nodosum Leprosum (ENL) skin lesions observed by fluorescence microscopy were found to contain immune complexes. While biopsies taken from at least seven ENL lesions contained deposits of immunoglobulin in their vessel walls, twelve of the lesions contained such deposits in the perivascular cell infiltrate. However, immunoglobulin deposits were observed in the dermis of all 25 ENL lesions studied. On the basis of localization of these fluorescent deposits the 25 skin lesions were classified under groups I to III and were correlated with the onset of the reaction. Control biopsies taken from the lepromatous leprosy patients without ENL were uniformly negative for immunoglobulin deposits.