CC chemokines and the receptors CCR3 and CCR5 are differentially expressed in the nonneoplastic leukocytic infiltrates of Hodgkin disease

Lymph nodes with Hodgkin disease (HD) harbor few neoplastic cells in a marked leukocytic infiltrate. Since chemokines are likely to be involved in the recruitment of these leukocytes, the expression of potentially relevant chemokines and chemokine receptors were studied in lymph nodes from 24 patients with HD and in 5 control lymph nodes. The expression of regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta was analyzed by in situ hybridization and that of CCR3 and CCR5 by immunohistochemistry and flow cytometry. It was found that, overall, the expression of all 4 chemokines was markedly enhanced, but the cellular source was different. RANTES was expressed almost exclusively by T cells whereas the expression of MCP-1, MIP-1alpha, and MIP-1beta was confined largely to macrophages. In control lymph nodes, chemokine expression was low, with the exception of MIP-1alpha in macrophages. CCR3 and CCR5 were highly expressed in T cells of HD involved but not of control lymph nodes. CCR3 was equally distributed in CD4+ and CD8+ cells, but CCR5 was associated largely with CD4+ cells. In HD lymph nodes, CCR3 and CCR5 were also expressed in B cells, which normally do not express these receptors. All these chemokines and receptors studied, by contrast, were absent in the neoplastic cells. It was concluded that chemokines are involved in the formation of the HD nonneoplastic leukocytic infiltrate. Expression of CCR3 and CCR5 appears to be characteristic of HD, but the roles of these receptors' up-regulation for the disease process remain unclear.

CC-chemokine receptors: a potential therapeutic target for Trypanosoma cruzi-elicited myocarditis

The comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate new therapeutic strategies aiming to ameliorate the inflammation that leads to heart dysfunction, without hampering parasite control. The augmented expression of CCL5/RANTES and CCL3/MIP-1alpha, and their receptor CCR5, in the heart of T. cruzi-infected mice suggests a role for CC-chemokines and their receptors in the pathogenesis of T. cruzi-elicited myocarditis. Herein, we discuss our recent results using a CC-chemokine receptor inhibitor (Met-RANTES), showing the participation of CC-chemokines in T. cruzi infection and unraveling CC-chemokine receptors as an attractive therapeutic target for further evaluation in Chagas disease.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.

The chemokines CCL11, CCL20, CCL21, and CCL24 are preferentially expressed in polarized human secondary lymphoid follicles

Chemokines regulate cellular trafficking to and from lymphoid follicles. Here, the distribution pattern of four CCL chemokines is defined by in situ hybridization in human lymphoid follicles from tonsils and lymph nodes (LNs) of newborns and adults. Cells expressing CCL11 (eotaxin) and CCL20 (Exodus) were preferentially located within follicles, while cells expressing CCL21 (secondary lymphoid-tissue chemokine) and CCL24 (eotaxin-2) mRNA were almost exclusively found in the perifollicular areas. Hence, the two CCR3-binding chemokines, CCL11 and CCL24, showed a mutually exclusive expression pattern in the intra- and extra-follicular areas, respectively. Chemokine gene expression paralleled follicular maturation: in tonsils, where approximately 80% of follicles are polarized, CCL11 and CCL20 mRNA-positive cells were detected more frequently than in lymph nodes from adults, where about half of follicles are non-polarized. No intrafollicular chemokine expression was detectable in the primary follicles from newborns. Extrafollicular cells expressing CCL21 and CCL24 were again more frequent in tonsils than in LNs from adults. The observed preferential presence of cells expressing CC chemokines in polarized human lymphoid follicles indicates that chemokines are not only instrumental in the induction of follicle formation, but may also be involved in their further differentiation.

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

Specific Activation of Leukocyte β2 Integrins Lymphocyte Function–associated Antigen-1 and Mac-1 by Chemokines Mediated by Distinct Pathways via the α Subunit Cytoplasmic Domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (αMβ2) but not lymphocyte function–associated antigen-1 (LFA-1; αLβ2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1α in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1α were confirmed by expression of αM or αL in αL-deficient Jurkat cells. Moreover, expression of chimeras containing αL and αM cytoplasmic domain exchanges indicated that α cytoplasmic tails conferred the specific mode of regulation. Coexpressing αM or chimeras in mutant Jurkat cells with a “gain of function” phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the αL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of β2 integrins. Our data suggest that a specific regulation of β2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the α subunit cytoplasmic domains.

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and - 3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P. J. Hudson. 2005. Nat. Biotechnol. 23: 1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways.

Paradoxically, morbid obesity was suggested to protect from cardiovascular co-morbidities as compared to overweight/obese patients. We hypothesise that this paradox could be inferred to modulation of the "endocannabinoid" system on systemic and subcutaneous adipose tissue (SAT) inflammation. We designed a translational project including clinical and in vitro studies at Geneva University Hospital. Morbid obese subjects (n=11) were submitted to gastric bypass surgery (GBS) and followed up for one year (post-GBS). Insulin resistance and circulating and SAT levels of endocannabinoids, adipocytokines and CC chemokines were assessed pre- and post-GBS and compared to a control group of normal and overweight subjects (CTL) (n=20). In vitro cultures with 3T3-L1 adipocytes were used to validate findings from clinical results. Morbid obese subjects had baseline lower insulin sensitivity and higher hs-CRP, leptin, CCL5 and anandamide (AEA) levels as compared to CTL. GBS induced a massive weight and fat mass loss, improved insulin sensitivity and lipid profile, decreased C-reactive protein, leptin, and CCL2 levels. In SAT, increased expression of resistin, CCL2, CCL5 and tumour necrosis factor and reduced MGLL were shown in morbid obese patients pre-GBS when compared to CTL. GBS increased all endocannabinoids and reduced adipocytokines and CC chemokines. In morbid obese SAT, inverse correlations independent of body mass index were shown between palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) levels and inflammatory molecules. In vitro, OEA inhibited CCL2 secretion from adipocytes via ERK1/2 activation. In conclusion, GBS was associated with relevant clinical, metabolic and inflammatory improvements, increasing endocannabinoid levels in SAT. OEA directly reduced CCL2 secretion via ERK1/2 activation in adipocytes.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

Immunopathogenèse de la cryptococcose chez la souris transgénique exprimant le génome du VIH-1

La cryptococcose chez les patients atteints du VIH-1 est principalement causée par Cryptococcus neoformans var. grubii tandis que Cryptococcus gattii infecte surtout les personnes immunocompétentes. Afin d’élucider les mécanismes causant la susceptibilité différentielle à l’égard de ces deux espèces de Cryptococcus dans le contexte de l’infection au VIH-1, nous avons utilisé un modèle novateur de la cryptococcose chez la souris transgénique CD4C/HIVMutA, qui exprime les gènes nef, env et rev du VIH-1. L’expression du transgène VIH-1 a augmenté le recrutement pulmonaire des macrophages alvéolaires mais a diminué celui des lymphocytes T CD4+ et CD8+ en réponse à l’infection par le C. neoformans ou le C. gattii. La production pulmonaire des chimiokines MCP-1 (CCL2) et RANTES (CCL5) était également réduite chez les souris transgéniques infectées par l’une ou l’autre de ces espèces de Cryptococcus. La production pulmonaire de MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 et IL-13 était augmentée chez la souris infectée au C. neoformans comparativement à C. gattii. In vitro, les macrophages alvéolaires prélevés chez la souris Tg et stimulés par des agonistes ont produit davantage de MIP-1β, alors que les chimiokines MCP-1 et RANTES n’ont pas été détectées.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Abstract Background Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.

THE ROLE OF MAP KINASES ON THE FUNCTIONAL HETEROGENEITY OF HUMAN CD8+ T CELL MATURATION SUBSETS

While prior studies have focused on naïve (CD45RA+CD27+) and early stage memory (CD45RA-CD27+) CD8+ T cells, late memory CD8+ T cells (CD45RA+CD27) have received less interest because this subset of T cells is generally recognized as effectors, which produce IFNγ (but no IL-2) and perforin. However, multiple studies suggest that late memory CD8+ T cells may provide inadequate protection in infectious diseases and cancer models. To better understand the unique function of late memory CD8+ T cells, I optimized multi-color flow cytometry techniques to assess the cytokine production of each human CD8+ T cell maturation subset. I demonstrated that late memory CD8+ T cells are the predominant producer of CC chemokines (e.g. MIP-1β), but rarely produce IL-2; therefore they do not co-produce IL-2/IFNγ (polyfunctionality), which has been shown to be critical for protective immunity against chronic viral infection. These data suggest that late memory CD8+ T cells are not just cytotoxic effectors, but may have unique functional properties. Determining the molecular signature of each CD8+ T cell maturation subset will help characterize the role of late memory CD8+ T cells. Prior studies suggest that ERK1 and ERK2 play a role in cytokine production including IL-2 in T cells. Therefore, I tested whether differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets contributes to their functional signature by a novel flow cytometry technique. I found that the expression of total ERK1, but not ERK2, is significantly diminished in late memory CD8+ T cells and that ERK1 expression is strongly associated with IL-2 production and CD28 expression. I also found that IL-2 production is increased in late memory CD8+ T cells by over-expressing ERK1. Collectively, these data suggest that ERK1 is required for IL-2 production in human CD8+ T cells. In summary, this dissertation demonstrated that ERK1 is down-regulated in human late memory CD8+ T cells, leading to decreased production of IL-2. The data in this dissertation also suggested that the functional heterogeneity in human CD8+ T cell maturation subsets results from their differential ERK1 expression.

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-Å resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.