Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1α and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4⁺ T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4⁺ T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-γ and ΤΝF-α in TB PF and their positive correlation with IP-10 and MIP-1α indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.     

Compartmentalization of a CD4+ T lymphocyte subpopulation in tuberculous pleuritis

The study of T lymphocytes from pleural fluid and tissue of patients with tuberculous pleuritis provides an opportunity to evaluate the human immune response to infection at the site of disease activity. Therefore, we investigated the phenotype and function of CD4+ pleural fluid cells from patients with tuberculous pleuritis. Pleural fluid was enriched with CD4+CDw29+ T lymphocytes, which are thought to represent "memory" T cells. Immunoperoxidase staining of pleural tissue confirmed the predominance of CD4+CDw29+ T lymphocytes at the site of disease activity. CD4+ subpopulations were evaluated for their ability to contribute to a cell-mediated immune response against Mycobacterium tuberculosis by assaying immune function in vitro. Pleural fluid-derived CD4+CDw29+ cells, but not CD4+CDw29- lymphocytes, proliferated vigorously and produced high levels of IFN-gamma when stimulated with purified protein derivative of M. tuberculosis. CD4+CDw29+ clones produced IFN-gamma specifically in response to purified protein derivative of M. tuberculosis but not to an irrelevant Ag, tetanus toxoid. IFN-gamma levels were markedly elevated in pleural fluid, compared to peripheral blood, suggesting production of this lymphokine in vivo at the site of tissue inflammation. The sum of these data indicate that, in tuberculous pleuritis, CD4+CDw29+ cells are concentrated at the site of disease activity, produce IFN-gamma and are likely to play an important role in the local human cell-mediated immune response to M. tuberculosis.     

Adenosine deaminase activity is a sensitive marker for the diagnosis of tuberculous pleuritis in patients with very low CD4 counts

Background

Adenosine Deaminase Activity (ADA) is a commonly used marker for the diagnosis of tuberculous pleural effusion. There has been concern about its usefulness in immunocompromised patients, especially HIV positive patients with very low CD4 counts. The objective of this study was to evaluate the sensitivity of ADA in pleural fluid in patients with low CD4 counts.

Materials and Methods

This was a retrospective case control study. Medical files of patients with tuberculous pleuritis and non-tuberculous pleuritis were reviewed. Clinical characteristics, CD4 cell counts in blood and biochemical markers in pleural fluid, including ADA were recorded.

Results

One ninety seven tuberculous pleuritis and 40 non- tuberculous pleuritis patients were evaluated. Using the cut-off value of 30 U/L, the overall sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of ADA was 94%, 95%, 19, and 0.06 respectively. The mean CD4 cell counts among TB pleuritis patients was 29 and 153 cells/microL in patients with CD4 <50 cells/microL and >50 cells/microL, (p<0.05) respectively. The corresponding mean ADA values for these patients were 76 U/L and 72 U/L respectively (p>0.5). There was no correlation between ADA values and CD4 cell counts (r = −0.120, p = 0.369).

Conclusion

ADA analysis is a sensitive marker of tuberculous pleuritis even in HIV patients with very low CD4 counts in a high TB endemic region. The ADA assay is inexpensive, rapid, and simple to perform and is of great value for the immediate diagnosis of tuberculous pleuritis while waiting for culture result and this has a positive impact on patient outcome.     

Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-producing CD56bright cells in tuberculous pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.     

Identification of M. tuberculosis-specific Th1 cells expressing CD69 generated in vivo in pleural fluid cells from patients with tuberculous pleurisy

Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M. tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4(+) T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4(+)CD69(+) T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4(+)CD69(+) T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4(+)CD69(-) T cells, demonstrating that CD4(+)CD69(+) T cells were MTB-specific Th1 cells. In addition, CD4(+)CD69(+) T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA(-)CCR7(-)CD62L(-)CD27(-)). Moreover, the percentages of CD4(+)CD69(+) T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4(+)CD69(+) but not CD4(+)CD69(-) fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4(+)CD25(+) Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4(+) T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Th17 and Th22 cells in psoriatic arthritis and psoriasis

Introduction

The aim of this study was to characterize interleukin 17 (IL-17) and interleukin 22 (IL-22) producing cells in peripheral blood (PB), skin, synovial fluid (SF) and synovial tissue (ST) in patients with psoriasis (Ps) and psoriatic arthritis (PsA).

Methods

Flow cytometry was used to enumerate cells making IL-22 and IL-17, in skin and/or SF and PB from 11 patients with Ps and 12 patients with PsA; skin and PB of 15 healthy controls and SF from rheumatoid arthritis (RA) patients were used as controls. Expression of the interleukin 23 receptor (IL-23R) and chemokine receptors CCR4 and CCR6 was examined. Secretion of IL-17 and IL-22 was measured by ELISA. ST was analysed by immunohistochemical staining of IL-17 and IL-22.

Results

Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were seen in PB of patients with PsA and Ps. IL-17 secretion was significantly elevated in both PsA and Ps, whilst IL-22 secretion was higher in PsA compared to Ps and healthy controls. A higher proportion of the CD4+ cells making IL-17 or IL-22 expressed IL-23R and frequencies of IL-17+, CCR6+ and CCR4+ T cells were elevated in patients with Ps and those with PsA. In patients with PsA, CCR6+ and IL-23R + T cells numbers were elevated in SF compared to PB. Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were demonstrated in Ps skin lesions. In contrast, whilst elevated frequencies of CD4+ IL-17+ cells were seen in PsA SF compared to PB, frequencies of CD4+ IL-22+ T cells were lower. Whereas IL-17 expression was equivalent in PsA, osteoarthritis (OA) and RA ST, IL-22 expression was higher in RA than either OA or PsA ST, in which IL-22 was strikingly absent.

Conclusions

Elevated frequencies of IL-17 and IL-22 producing CD4+ T cells were a feature of both Ps and PsA. However their differing distribution at disease sites, including lower frequencies of IL-22+ CD4+ T cells in SF compared to skin and PB, and lack of IL-22 expression in ST suggests that Th17 and Th22 cells have common, as well as divergent roles in the pathogenesis of Ps and PsA.     

Differential susceptibility to staphylococcal superantigen (SsAg)-induced apoptosis of CD4+ T cells from atopic dermatitis patients and healthy subjects: the inhibitory effect of IL-4 on SsAg-induced apoptosis

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4(+) T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVbeta3(+) or Vbeta12(+) or Vbeta17(+)) CD4(+) T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3(+) cells in SEB-reactive CD4(+) T cells from healthy subjects and CCR4(+) cells in those from AD patients. SEB-reactive CD4(+) T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4(+) T cells. CXCR3(+) CD4(+) T cells were more susceptible to SEB-induced apoptosis than CCR4(+) CD4(+) T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4(+) and CXCR3(+) CD4(+) T cells but not of CCR4(+) CD4(+) T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4(+) T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.     

CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.     

Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimulated dendritic cells to differentiate into Th2 cells

Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.     

Increased frequency of CCR4+ and CCR6+ memory T-cells including CCR7+CD45RAmed very early memory cells in granulomatosis with polyangiitis (Wegener's)

Introduction

Chemokine receptors play an important role in mediating the recruitment of T cells to inflammatory sites. Previously, small proportions of circulating Th1-type CCR5+ and Th2-type CCR3+ cells have been shown in granulomatosis with polyangiitis (GPA). Wondering to what extent CCR4 and CCR6 expression could also be implicated in T cell recruitment to inflamed sites in GPA, we investigated the expression of CCR4 and CCR6 on T cells and its association with T cell diversity and polarization.

Methods

Multicolor flow cytometry was used to analyze CCR4, CCR6, and intracellular cytokine expression of T cells from whole blood of GPA-patients (n = 26) and healthy controls (n = 20). CCR7 and CD45RA were included for phenotypic characterization.

Results

We found a significant increase in the percentages of circulating CCR4+ and CCR6+ cells within the total CD4+ T cell population in GPA. In contrast, there was no difference in the percentages of CD8+CCR4+ and CD8+CCR6+ T cells between GPA and healthy controls. CCR4 and CCR6 expression was largely confined to central (T_CM) and effector memory T cells (T_EM, T_EMRA). A significant increase in the frequency of CCR4+ and CCR6+ T_EMRA and CCR6+ T_CM was shown in GPA. Of note, we could dissect CCR4 and CCR6 expressing CCR7+CD45RA^med very early memory T cells (T_VEM) from genuine CCR7+CD45RA^high naïve T cells lacking CCR4 and CCR6 expression for peripheral tissue-migration within the CCR7+CD45RA+ compartment. The frequencies of CCR4+ and CCR6+ T_VEM were also significantly increased in GPA. An increased percentage of IL-17+ and IL-22+ cells was detected in the CCR6+ cell subsets and IL-4+ cells in the CRR4+ cell subset when compared with CD4+ cells lacking CCR4 and CCR6 expression.

Conclusions

Increased frequencies of circulating CCR4+ and CCR6+ memory T cell subsets including hitherto unreported T_VEM suggest persistent T cell activation with the accumulation of CCR4+ and CCR6+ cells in GPA. CCR4 and CCR6 could be involved in the recruitment of T cells including cytokine-producing subsets to inflamed sites in GPA.     

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.