Evaluation of localized and systemic immune responses in cutaneous leishmaniasis caused by Leishmania tropica: interleukin-8, monocyte chemotactic protein-1 and nitric oxide are major regulatory factors

Abstract

We have established Leishmania tropica as the causative agent of cutaneous leishmaniasis (CL) in the region of India where the disease is endemic. The association between localized and circulating levels of immune-determinants in CL patients was evaluated. Reverse transcription-polymerase chain reaction analysis revealed up-regulation of interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha (TNF-alpha), IL-10 and IL-4 in dermal lesions at the pretreatment stage (n = 31) compared with healthy controls (P < 0.001) and a significant down-regulation after treatment (n = 14, P < 0.05). The results indicated that an unfavourable clinical outcome in CL was not related to an inadequate T helper 1 (Th1) cell response, but rather to impairment in multiple immune functions. Comparative assessment of treatment regimes with rifampicin (RFM) or sodium antimony gluconate (SAG) revealed tissue cytokine levels to be significantly reduced after treatment with RFM (P < 0.005), while no significant decrease was evident in the levels of IFN-gamma, TNF-alpha and IL-10 (P > 0.05) as a result of treatment with SAG. Increased transcripts of monocyte chemoattractant protein-1 (MCP-1) (P < 0.001) and inducible nitric oxide synthase (iNOS) (P < 0.05) were evident before treatment in tissue lesions and remained high after treatment. Immunohistochemistry demonstrated strong expression of myeloperoxidase (MPO) and IL-8, and moderate expression of iNOS in dermal lesions. The expression levels of IL-8, MCP-1 and nitric oxide (NO) were high in patient sera before treatment, as determined using cytokine bead array and enzyme-linked immunosorbent assay (ELISA). At the post-treatment stage, the serum IL-8 levels had decreased; however, the levels of MCP-1 and NO remained high. These data suggest that IL-8 is an effector immune-determinant in the progression of CL, whereas NO facilitates the parasite killing by macrophages via MCP-1-mediated stimulation.

Figure 1

Analysis of intralesional cytokines in cutaneous leishmaniasis (CL) patients at the pretreatment stage. Levels of expression of messenger RNA (mRNA) for interleukin (IL)-1β, -4, -8 and -10, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in dermal lesions of CL patients at the pretreatment stage (n = 31) and in healthy controls (n = 6). Normalized complementary DNA (cDNA) was amplified using respective of cytokine primers. Polymerase chain reaction products were electrophoresed, and the intensity of the signal was determined by densitometry. The graph shows the results as an expression index, defined as the ratio of the intensity of cytokine with respect to the hypoxanthine phosphoribosyltransferase (HPRT) gene. The bars indicate standard errors (SEs). P<0·001 was statistically significant.

Figure 2

Comparative analysis of intralesional immunodeterminants in cutaneous leishmaniasis (CL) patients at pretreatment and post-treatment stages. (a) Levels of expression of messenger RNA (mRNA) for interleukin (IL)-1β, -4, -8 and -10, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), monocyte chemotactic factor-1 (MCP-1) and inducible nitric oxide synthase (iNOS) in paired lesion samples from CL patients pretreatment (n = 9) and post-treatment (n = 9), and in healthy controls (n = 6). (b) Levels of cytokine transcripts in lesion tissues from CL patients at the pretreatment stage (n = 31) and after treatment with sodium stibogluconate (SAG, n = 6) or rifampicin (RFM, n = 7). Normalized complementary DNA (cDNA) was amplified using the respective cytokine primers. Other details are the same as in Fig. 1.

Figure 3

Quantitative estimation of interleukin-8 (IL-8) in the sera of cutaneous leishmaniasis (CL) patients using cytokine bead array analysis (CBA). Analysis of IL-8 at the protein level was carried out at pretreatment (n = 15) and post-treatment (n = 9) stages, and in healthy controls (n = 4). Values are mean ± standard error (SE). The bars indicate SEs.

Figure 4

Quantitative enzyme-linked immunosorbent assay (ELISA) analysis of (a) interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) and (b) nitric oxide (NO) in the sera of patients. (a) Levels (pg/ml) of IL-8 and MCP-1, in a 1 : 20 dilution of serum, at pretreatment (n = 20), and post-treatment (n = 9) stages in patients and controls (n = 9). (b) Serum NO level (μm) at pretreatment (n = 32) and post-treatment (n = 10) stages in Indian cutaneous leishmaniasis (CL) patients and controls (n = 10). Values are mean ± standard error (SE). The bars indicate SEs.

Figure 5

Immunohistochemical analysis of tissue biopsies from cutaneous leishmaniasis (CL) patients for myeloperoxidase (a), interleukin-8 (IL-8) (c) and inducible nitric oxide synthase (iNOS) (e), and in the respective controls (b, d, f ). Immunostaining in tissue sections was visualized at a magnification of × 20.