Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies.

The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article reviews basic mechanisms in the recruitment of leukocytes to tissues and then selectively reviews new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. Investigators have also begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise.

CCL19 is a specific ligand of the constitutively recycling atypical human chemokine receptor CRAM-B.

The human chemokine receptor CRAM (chemokine receptor on activated macrophages), encoded by the gene CCRL2, is a new candidate for the atypical chemokine receptor family that includes the receptors DARC, D6 and chemocentryx chemokine receptor (CCX-CKR). CRAM is maturation-stage-dependently expressed on human B lymphocytes and its surface expression is up-regulated upon short-term CCL5 exposure. Here, we demonstrate that the homeostatic chemokine CCL19 is a specific ligand for CRAM. In radioactive labelling studies CCL19 bound to CRAM-expressing cells with an affinity similar to the described binding of its other receptor CCR7. In contrast to the known CCL19/CCR7 ligand/receptor pair, CRAM stimulation by CCL19 did not result in typical chemokine-receptor-dependent cellular activation like calcium mobilization or migration. Instead, we demonstrate that CRAM is constitutively recycling via clathrin-coated pits and able to internalize CCL19 as well as anti-CRAM antibodies. As this absence of classical chemokine receptor responses and the recycling and internalization features are characteristic for non-classical chemokine receptors, we suggest that CRAM is the newest member of this group. As CCL19 is known to be critically involved in lymphocyte and dendritic cell trafficking, CCL19-binding competition by CRAM might be involved in modulating these processes.

Role of the atypical chemoattractant receptor CRAM in regulating CCL19 induced CCR7 responses in B-cell chronic lymphocytic leukemia.

BACKGROUND: The non-signalling chemokine receptors, including receptors DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM (also known as HCR or CCRL2) is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner and absent on T cells. We have recently shown that it competitively binds CCL19. CCL19 and its signalling receptor CCR7 are critical components involved in cell recruitment to secondary lymphoid organs and in maturation. B cell Chronic Lymphocytic Leukemia (B-CLL) is a low-grade lymphoma characterized by proliferative centres (or pseudofollicles). Proliferative centres develop due to abnormal cellular localisation and they are involved in the development of malignant cells. CCR7 is highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21, while CRAM expression and potential interferences with CCR7 are yet to be characterized. RESULTS: In this study, we show that B cells from patients with B-CLL present highly variable degrees of CRAM expression in contrast to more consistently high levels of CCR7. We investigated the hypothesis that, similar to the atypical receptor DARC, CRAM can modulate chemokine availability and/or efficacy, resulting in the regulation of cellular activation. We found that a high level of CRAM expression was detrimental to efficient chemotaxis with CCL19. MAP-kinase phosphorylation and intracellular calcium release induced by CCL19 were also altered by CRAM expression. In addition, we demonstrate that CRAM-induced regulation of CCL19 signalling is maintained over time. CONCLUSIONS: We postulate that CRAM is a factor involved in the fine tuning/control of CCR7/CCL19 mediated responses. This regulation could be critical to the pivotal role of CCL19 induced formation of proliferation centres supporting the T/B cells encounter as well as disease progression in B-CLL.

The role of chemokines in B cell chronic lymphocytic leukaemia: pathophysiological aspects and clinical impact.

Chemokines are centrally involved in leukocyte migration, homing and haematopoiesis. Besides these physiological aspects, their role in pathological processes especially with respect to solid tumour and haematological neoplasias is well established. In this context, the focus was set here on disclosing their contribution in B cell chronic lymphocytic leukaemia (B-CLL), which is regarded as the most characteristic low-grade lymphoma. Up to now, it has been demonstrated that several chemokines are involved in migration of B-CLL cells to lymph nodes, secondary lymphoid organs and bone marrow. Moreover, some chemokines are known to have an anti-apoptotic effect and thus contribute to the survival of B-CLL cells. By interfering with both of these aspects, new therapeutic targets for this yet incurable disease may be developed. Furthermore, a correlation can be drawn between the concentration of some chemokines in patients' serum, the expression of their respective receptors on B-CLL cells and well-established predictive clinical parameters. Consequently, further systematic investigation of the chemokine network may lead to the identification of new diagnostic and prognostic markers. This review focuses on the impact of chemokines and their receptors on B-CLL pathophysiology and points out potential implications for both treatment and diagnosis.

Human B cells express the orphan chemokine receptor CRAM-A/B in a maturation-stage-dependent and CCL5-modulated manner.

Chemokines orchestrate the organization of leucocyte recruitment during inflammation and homeostasis. Despite growing knowledge of chemokine receptors, some orphan chemokine receptors are still not characterized. The gene CCRL2 encodes such a receptor that exists in two splice variants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by human peripheral blood and bone marrow B cells, and by different B-cell lines dependent on the B-cell maturation stage. Intriguingly, CRAM surface expression on the pre-B-cell lines Nalm6 and G2 is specifically upregulated in response to the inflammatory chemokine CCL5 (RANTES), a chemokine that is well known to play an important role in modulating immune responses. Although Nalm6 cells do not express any of the known CCL5 binding receptors, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a direct effect of CCL5 through the CRAM receptor. However, no calcium mobilization or migratory responses upon CCL5 stimulation are induced in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation cannot be inhibited by pertussis toxin, suggesting that CRAM does not couple to Gi proteins. Our results describe the expression of a novel, non-classical chemokine receptor on B cells that is potentially involved in immunomodulatory functions together with CCL5.

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton.

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

Introduction for the special issue on "Tissue Barriers in Inflammation".

This issue of Tissue Barriers contains the inaugural special issue devoted to recent advances in barrier function of endothelial and epithelial cells. We used this opportunity to invite experts in vascular endothelial cell biology and epithelial cell biology to comment on critical questions and problems in permeability of organ and tissue barriers, and to provide insight into common areas in these fields, namely how these cells maintain homeostasis and response to injury and infection. To complement these reviews, this issue also contains four research articles that explore specific questions related respiratory and intestinal epithelial cell function.