Cytokine directed therapy in scleroderma: rationale,current status,and the future

The hallmark of scleroderma is cutaneous and visceral fibrosis characterized and by increased biosynthesis of multiple matrix proteins by interstitial fibroblasts. Studies over recent years have delineated pathways involved in promoting matrix synthesis and elucidated the molecular pathways of regulation. Central to the regulation of fibrosis are extracellular mediators, called cytokines, which are elaborated by a variety of cells, including those in the immune system, vascular cells, and fibroblasts themselves. The concept that inhibiting or promoting the action of these naturally occurring profibrotic or antifibrotic molecules, respectively, is a rational therapeutic approach to treating scleroderma and other fibrotic diseases finds support in animal studies and anticytokine therapy conducted in relation to rheumatoid arthritis and other disorders. This review looks at cytokines known or thought to play a role in scleroderma and/or other fibrotic states and at potential therapy directed at these mediators. Potential targets for therapy include transforming growth factor beta (TGF-beta), connective tissue growth factor (CTGF), IL-4, IL-13, MCP-1, and endothelin, among others.     

The role of chemokines in the pathogenesis of scleroderma

PURPOSE OF REVIEW: The triad of pathologic changes that defines systemic sclerosis (scleroderma) includes immune system activation with autoimmunity; an obliterative, proliferative small vessel vasculopathy; and fibrosis. Available data suggest that several cytokines, including chemokines, contribute to the development of scleroderma complications. This review focuses on chemokines and their contribution to tissue fibrosis and pulmonary hypertension in scleroderma. RECENT FINDINGS: Proteins and mRNAs for monocyte chemoattractant protein-1; pulmonary and activation-regulated chemokine; macrophage inflammatory protein-1, regulated upon activation normal T cell expressed and secreted; interleukin-8; and transforming growth factor-beta have been found in increased amounts in blood or involved tissue from scleroderma patients. These factors are likely to contribute directly to tissue damage in scleroderma through several pathways, including stimulation of extracellular matrix production, induction of TGF-beta production and activation, and chemoattraction of T cells and nonspecific inflammatory cells into tissues. SUMMARY: Multiple chemokines are part of the pathologic network that causes tissue damage in scleroderma, and, as such, may provide therapeutic targets in scleroderma.     

Requirement of CCL17 for CCR7- and CXCR4-dependent migration of cutaneous dendritic cells

Chemokines are known to regulate the steady-state and inflammatory migration of cutaneous dendritic cells (DCs). The β-chemokine CCL17, a ligand of CCR4, is inducibly expressed in a subset of DCs and is strongly up-regulated in atopic diseases. Using an atopic dermatitis model, we show that CCL17-deficient mice develop acanthosis as WT mice, whereas dermal inflammation, T helper 2-type cytokine production, and the allergen-specific humoral immune response are significantly decreased. Notably, CCL17-deficient mice retained Langerhans cells (LCs) in the lesional skin after chronic allergen exposure, whereas most LCs emigrated from the epidermis of allergen-treated WT controls into draining lymph nodes (LNs). Moreover, CCL17-deficient LCs showed impaired emigration from the skin after exposure to a contact sensitizer. In contrast, the absence of CCR4 had no effect on cutaneous DC migration and development of atopic dermatitis symptoms. As an explanation for the major migratory defect of CCL17-deficient DCs in vivo, we demonstrate impaired mobility of CCL17-deficient DCs to CCL19/21 in 3D in vitro migration assays and a blockade of intracellular calcium release in response to CCR7 ligands. In addition, responsiveness of CCL17-deficient DCs to CXCL12 was impaired as well. We demonstrate that the inducible chemokine CCL17 sensitizes DCs for CCR7- and CXCR4-dependent migration to LN-associated homeostatic chemokines under inflammatory conditions and thus plays an important role in cutaneous DC migration.     

Short communication: persistence of high blood levels of the chemokines CCL2, CCL19, and CCL20 during the course of HIV infection

Dendritic cells (DCs) are important mediators of the immune response against HIV and yet blood DC numbers fall substantially during HIV infection. Here we report that blood levels of the DC-tissue tropic chemokines monocyte chemotactic protein (MCP-1/CCL2), macrophage inflammatory proteins (MIP-3α/CCL20), and MIP-3β/CCL19 remained elevated throughout the course of HIV infection suggesting that the relatively low levels of blood circulating DCs may be due to active recruitment of these cells to peripheral sites to fight disease progression.     

The role of chemokines in controlling granulomatous inflammation in Schistosoma mansoni infection

Chemokines are a superfamily of low-molecular-weight cytokines that were initially described for their chemoattractant activity. It is now clear chemokines have several other activities that modulate immune processes. Chemokines appear to play a role in the pathogenesis of several inflammatory diseases. The role of chemokines and their receptors in mediating granulomatous inflammation induced by Schistosoma mansoni egg antigens presented in particulate manner have been studied in detail. Much less is known of the role of chemokines in mediating inflammation during the course of S. mansoni infection. Our studies in mice suggest a relevant role for the chemokine CCL3 and the receptor CCR5 in the pathogenesis of experimental S. mansoni infection. Absence of CCL3 is associated with decrease in granuloma size, fibrosis and parasite load. In humans, levels of CCL3 in plasma associate with disease severity and may be useful for diagnostic purposes. In contrast, absence of CCR5 is associated with enhanced lethality, granuloma size and fibrosis. It is suggested that the balance of chemokine production and chemokine receptor activation are important determinants of the fate of infection in experimental animals and humans.     

CCL22 and CCL17 in rat radiation pneumonitis and in human idiopathic pulmonary fibrosis.

Pulmonary fibrosis is caused by various known and unknown aetiologies, but the key pathogenic mechanisms are still ill-defined. Chemokines are a large family of chemotactic cytokines that play pivotal roles in various inflammatory diseases. In the present study, the roles of chemokines in a rat model of radiation pneumonitis/ pulmonary fibrosis were examined. Accumulation of inflammatory cells and pneumonitis were observed on day 28, and diffuse alveolar wall thickening with extensive fibrosis was observed on day 56. In addition to the previously reported CCL2 (macrophage chemoattractant protein-1) induction, selective upregulation of CCL22 (macrophage-derived chemokine) and CCL17 (thymus and activation-regulated chemokine) were demonstrated for the first time in the irradiated lung tissues. Immunohistochemically, it was demonstrated that CCL22 and CCL17 were localised primarily to alveolar macrophages, whereas their receptor CC chemokine receptor 4 (CCR4) was detected on alveolar lymphocytes and macrophages. On further analysis of bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis and sarcoidosis, elevated levels of CCL22, but not of CCL17, were observed in the idiopathic pulmonary fibrosis patients. Since these two chemokines play pivotal roles in various type-2 T-helper cell-dominant diseases, it was speculated that CCL22, and probably CCL17, are involved in the pathophysiology of radiation pneumonitis/pulmonary fibrosis and idiopathic pulmonary fibrosis through the recruitment of CC chemokine receptor 4-positive type-2 T-helper cells and alveolar macrophages.     

Serum chemokine CXC ligand 10 (CXCL10) predicts fibrosis progression after liver transplantation for hepatitis C infection

The recurrence of liver fibrosis after liver transplantation (LT) for hepatitis C virus (HCV) infection is responsible for graft loss and patient mortality. Although the contribution of the immune system to fibrosis recurrence is anticipated, systematic studies evaluating immune parameters as predictive markers of allograft fibrosis are lacking. The infiltration of immune cells into the graft is governed by chemokines. Here we assessed the predictive value of serum levels of chemokines [chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11, and chemokine (C-C motif) ligand 2 (CCL2)] with respect to fibrosis recurrence after LT in 90 HCV-infected organ recipients. Chemokines were determined within the first and third years after LT and were correlated with histological fibrosis progression in protocol biopsy samples at 1, 3, 5, and 7 years (median follow-up = 3 years). The association of chemokines with fibrosis progression was assessed by univariate and multivariate analyses and by Cox regression analysis. The results for the analyzed chemokines showed that CXCL10 levels in the first year after LT were strongly associated with early fibrosis recurrence (P = 0.005) independently of risk confounders (including the donor age, HCV viral load, HCV genotype, acute rejection, and inflammatory activity). As assessed by Cox regression analysis, a CXCL10 serum level ≤ 140 pg/mL was significantly predictive of the absence of F2 fibrosis (P = 0.001), whereas a level ≤ 220 pg/mL early after LT predicted the absence of F3 fibrosis during follow-up (P = 0.035). CONCLUSION: CXCL10 is an independent biomarker of the recurrence of significant fibrosis after LT for HCV infection. These results might guide patients' care after transplantation and help us to select optimal candidates for antiviral therapy post-LT.     

New chemokine targets for asthma therapy

Chemokines and chemokine receptors are part of a complex network of molecules that play a key role in leukocyte migration and activation. The chemokine family role is crucial in the immune system, orchestrating innate and acquired immune responses, but also in allergic inflammation. A subset of chemokines, including CCL11, CCL24, CCL26, CCL7, CCL13, CCL17, and CCL22 is highly expressed by the three main cell types involved in allergic inflammation: eosinophils, basophils, and Th2 lymphocytes. In vitro and in vivo experimental studies in murine models of asthma as well as evidence from patients with asthma confirm the role of these chemokines and their receptors, including CCR3, CCR4, and CCR8, establishing a subset of chemokine/chemokine receptor that is potentially important in allergic inflammation. Recent data support the concept that interfering with chemokines or chemokine receptors represents a new approach in allergy therapy. However, even if some of them have been shown to be effective in animal models, none is as yet used in human patients.     

Cytokines as therapeutic targets for the gastrointestinal manifestations of scleroderma.

Systemic sclerosis (SSc), or scleroderma, is a connective tissue disorder characterized by progressive fibrosis of the skin and internal organs. It has significance for gastroenterologists because the gastrointestinal tract is involved in 90% of SSc patients, who often present with esophageal dysfunction. Though the exact pathogenesis of SSc is unknown, there is increasing evidence supporting an immune mechanism. Cytokines are the soluble mediators of immune activation, altered fibroblast proliferation and extracellular matrix accumulation in SSc and thereby provide important therapeutic targets. In the present review, the involvement of cytokines in SSc is discussed with particular emphasis on cytokines and growth factors that have been implicated in the disease process and likely play an important role in the gastrointestinal manifestations of scleroderma. The role of cytokines as therapeutic targets in scleroderma forms the basis of this timely review.     

Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages

Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE₂). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.     

Inflammatory cytokines modulate chemokine production patterns of HepG2 cells toward initially inclined direction

Aim:  Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations.
Methods:  HepG2 cells were stimulated with IL-1α, IFN-γ, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR.
Results:  (i) IL-1α up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-γ increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-γ-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-γ to the culture of IL-1α-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1α-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression.
Conclusions:  IFN-γ augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region.     

Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function

Trafficking of dendritic cells (DCs) to peripheral tissues and to secondary lymphoid organs depends on chemokines and lipid mediators. Here, we show that bone marrow-derived DCs (BM-DCs) express functional leukotriene B4 (LTB4) receptors as observed in dose-dependent chemotaxis and calcium mobilization responses. LTB4, at low concentrations, promoted the migration of immature and mature DCs to CCL19 and CCL21, which was associated with a rapid (30-minute) increase of CCR7 expression at the membrane level. At longer incubation times (6 hours), gene array analysis revealed a promoting role of LTB4, showing a significant increase of CCR7 and CCL19 mRNA levels. BM-DCs cultured from BLT1-/- or BLT1/2-/- mice showed a normal phenotype, but in vivo BLT1/2-/-DCs showed dramatic decrease in migration to the draining lymph nodes relative to wild-type (WT) DCs. Consistent with these observations, BLT1/2-/- mice showed a reduced response in a model of 2,4-dinitro-fluorobenzene (DNFB)-induced contact hypersensitivity. Adoptive transfer of 2,4-dinitrobenzene sulfonic acid (DNBS)-pulsed DCs directly implicated the defect in DC migration to lymph node with the defect in contact hypersensitivity. These results provide strong evidence for a role of LTB4 in regulating DC migration and the induction of adaptive immune responses.     

Endothelial induction of the T-cell chemokine CCL21 in T-cell autoimmune diseases

The signals that mediate T-cell infiltration during T-cell autoimmune diseases are poorly understood. The chemokine CCL21 (originally isolated by us and others as Exodus-2/6Ckine/SLC/TCA4) is highly potent and highly specific for stimulating T-cell migration. However, it is thought to be expressed only in secondary lymphoid organs, directing naive T cells to areas of antigen presentation. It is not thought to play a role in T-cell effector function during a normal immune response. In this study we tested the expression of T-cell chemokines and their receptors during T-cell autoimmune infiltrative skin diseases. By using immunohistology it was found that the expression of CCL21 but not CCL19 or 20 was highly induced in endothelial cells of T-cell autoimmune diseases. The receptor for CCL21, CCR7, was also found to be highly expressed on the infiltrating T cells, most of which expressed the memory CD45Ro phenotype. These data imply that the usual loss of CCL21 responsiveness in the normal development of memory T-cell effector function does not hold for autoimmune skin diseases.     

Chemokine networks in atopic dermatitis: traffic signals of disease

Atopic dermatitis is a chronic or chronically relapsing inflammatory skin disease with a prevalence ranging from 10% to 20% in children and 1% to 3% in adults of developed countries. Skin-infiltrating leukocytes play a pivotal role in the initiation and amplification of atopic skin inflammation. Recent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines. A subset of chemokines including CCL27, CCL17, CCL22, CCL18, CCL11, and CCL13 are highly expressed in atopic dermatitis. The corresponding chemokine receptors are found on the main leukocyte subsets involved in allergic skin inflammation, such as T cells, eosinophils, and dendritic cells. In this article, we provide an overview of the role of chemokines in the complex immunopathogenesis of atopic dermatitis, highlighting potential areas for therapeutic intervention.     

The role of mast cells in allergic inflammation

The histochemical characteristics of human basophils and tissue mast cells were described over a century ago by Paul Ehrlich. When mast cells are activated by an allergen that binds to serum IgE attached to their Fc?RI receptors, they release cytokines, eicosanoids and their secretory granules. Mast cells are now thought to exert critical proinflammatory functions, as well as potential immunoregulatory roles, in various immune disorders through the release of mediators such as histamine, leukotrienes, cytokines chemokines, and neutral proteases (chymase and tryptase). The aim of this review is to describe the role of mast cells in allergic inflammation. Mast cells interact directly with bacteria and appear to play a vital role in host defense against pathogens. Drugs, such as glucocorticoids, cyclosporine and cromolyn have been shown to have inhibitory effects on mast cell degranulation and mediator release. This review shows that mast cells play an active role in such diverse diseases as asthma, rhinitis, middle ear infection, and pulmonary fibrosis. In conclusion, mast cells may not only contribute to the chronic airway inflammatory response, remodeling and symptomatology, but they may also have a central role in the initiation of the allergic immune response, that is providing signals inducing IgE synthesis by B-lymphocytes and inducing Th2 lymphocyte differentiation.     

CCR5 expression and CC chemokine levels in idiopathic pulmonary fibrosis.

CC chemokines play an important role in the pathogenetic mechanisms of interstitial lung disease, while a downregulation of CC chemokine receptor (CCR)5 in the fibrotic stages of sarcoidosis has been observed. To evaluate the involvement of CC chemokines and the expression of CCR5 in idiopathic pulmonary fibrosis (IPF) and, more specifically, in usual interstitial pneumonia, 35 subjects were studied. CC chemokine ligand (CCL)2, CCL3 and CCL4 levels were measured in the bronchoalveolar lavage fluid (BALF) of 18 nonsmoker control subjects and 17 patients affected by IPF. CCR5 expression was evaluated in alveolar macrophages and lymphocytes. The BALF levels of all chemokines were significantly increased in IPF: median (range) CCL3 1.6 (1.0-11.1) versus 1.2 (0.0-3.8) pg x mL(-1); CCL4 6.2 (1.3-96.0) versus 3.4 (0.3-6.8) pg x mL(-1); and CCL2 60.1 (16.7-251.3) versus 4.6 (0.5-119.4) pg x mL(-1). CCL2 levels correlated negatively with the carbon monoxide diffusing capacity of the lung (D(L,CO)) and arterial oxygen tension. CCR5 expression was significantly reduced in lymphocytes from IPF compared with controls. The CC chemokines investigated are involved in the inflammatory mechanisms of idiopathic pulmonary fibrosis, and the results are in agreement with the hypothesis of a downregulation of the T-helper 1 immunological response in this disease.     

RANTES antagonism: a promising approach to treat chronic liver diseases

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl(4)) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5(-/-) mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.     

Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis

BACKGROUND: Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue. OBJECTIVE: To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention. METHODS: Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non-parametric tests were used for statistical analysis. RESULTS: Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen. DISCUSSION: A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1(+) and CCR5(+) cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.