Publicação da IMMUNITY aborda tema importante sobre o papel das quimiocinas na adesão de leucócitosPreview
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| Refers to: |  | Endothelial Heparan Sulfate Controls Chemokine Presentation in Recruitment of Lymphocytes and Dendritic Cells to Lymph Nodes Immunity, Volume 33, Issue 5, 24 November 2010, Pages 817-829, Xingfeng Bao, E. Ashley Moseman, HideoSaito, Bronislawa Petryanik, Aude Thiriot, Shingo Hatakeyama, Yuki Ito, Hiroto Kawashima, Yu Yamaguchi, John B. Lowe, Ulrich H. von Andrian, Minoru Fukuda |
In this issue of Immunity, Bao et al. (2010) provide in vivo evidence that heparan sulfate glycosaminoglycans (GAGs) are indispensable for immobilization and function of major chemokines required for leukocyte adhesion to and crossing through blood and lymphatic vessels.
Main Text
Chemokines are structurally related chemotactic cytokines (chemoattractants) with remarkable functional versatility (Bromley et al., 2008). Chemokines signal through cognate G protein coupled receptors (GPCRs) either at their soluble or immobilized states. However, a direct in vivo proof for their functions in soluble versus immobilized states has been difficult to obtain, because soluble chemokines are readily removed by conventional histological analysis. Thus, a genetic interference with chemokine immobilization has been necessary in order to dissect the significance of immobilization for particular adhesive and migratory processes.Leukocyte extravasation from blood involves sequential chemoattractant-mediated signals. Chemokines stably immobilized on surface proteoglycans on the luminal surface of endothelial cells were suggested to play a pivotal role in integrin-mediated arrest of rolling leukocytes (Ley et al., 2007). Additional chemokine signals were suggested to promote crawling of leukocytes across endothelium, protrusion, and encounter of abluminal chemokines (Ley et al., 2007). Chemokine immobilization on vascular endothelial cells and adherent platelets was suggested to be critical not only to prevent their dilution by blood flow but also to facilitate localized signaling to integrins on rolling leukocytes. In addition, stroma-immobilized chemokines efficiently promote motility of lymphocytes and dendritic cells (DCs) in specific areas of lymphoid tissues (Bajenoff et al., 2006). However, it is still unclear whether chemokines that direct leukocyte motility and chemotaxis in various interstitial spaces as well as across epithelial barriers operate in their soluble or immobilized states (Schumann et al., 2010).
Most chemokines share a carboxyl terminus stretch of positively charged residues that recognize heparan sulfate (HS) GAGs with moderate affinities ([Proudfoot, 2006] and [Rot and von Andrian, 2004]). HS GAGs are ubiquitous and structurally diverse macromolecules that interact with many cytokines, growth factors, and extracellular matrix (ECM) components. In vitro and in vivo studies on leukocyte interactions with various endothelial cells have suggested that many chemokines immobilize, and at times also oligomerize on, HS GAGs (Proudfoot, 2006). The first in vivo involvement of endothelial heparan sulfate in inflammation was genetically supported by elegant endothelial-targeted ablation of the enzyme required for N-sulfation of HS GAGs (Wang et al., 2005). Attenuated neutrophil infiltration to sites of inflammation was reported in these mice but was attributed to combined inhibition of chemokine transcytosis across endothelial cells, chemokine presentation on lumenal aspects of inflamed blood vessels, and to reduced expression of atypical endothelial L-selectin ligands necessary for optimal neutrophil rolling (Wang et al., 2005). The involvement of HS GAGs in chemokine immobilization on endothelial cells was demonstrated in that study only in vitro and so, direct in vivo evidence that HS GAGs immobilize endogenous chemokines has been missing. Such evidence has been especially necessary for lymphoid organ chemokines, because a major chemokine in this subgroup, the CCR7 ligand CCL21, was reported to also bind chondroitin sulfate GAGs ([Rot and von Andrian, 2004] and [Miyasaka and Tanaka, 2004]) as well as to ECM proteins, like collagen IV, and to a specialized scaffold, MAC25, within basement membranes of high endothelial venules (HEVs) (Miyasaka and Tanaka, 2004).
In this milestone in vivo study, Bao et al. (2010) temporally deleted the entire heparan sulfate glycans from all endothelial beds in a mouse model. The authors inactivated exostoses-1 (Ext1), a critical factor in early steps of heparan sulfate biosynthesis, with a drug-inducible endothelial specific promoter (Bao et al., 2010). Nearly total deletion of HS GAGs, achieved in the HEVs of skin draining and mesenteric lymph nodes, led to abolished presentation of CCL21 as evaluated by immunofluorescence staining. Accordingly, lymphocyte homing to lymph nodes was severely reduced and elegant intravital microscopy studies of inguinal lymph nodes indicated that this homing defect was due to loss of integrin activation on rolling lymphocytes and not to a defect in L-selectin-mediated rolling (Figure 1). The authors confirmed normal HEV architecture, together with retained expression of the endothelial marker PECAM-1, the LFA-1 integrin ligand, ICAM-1, and of MECA-79, a sulfated marker of HEV L-selectin ligands. Deletion of Ext1 also abolished HEV presentation of the inflammatory chemokine CCL2, transported from extralymphoid tissues (Bao et al., 2010).
| Full-size image (54K) High-quality image (405K) |
Figure 1.
Postulated Roles for Endothelial HS Proteoglycans in Chemokine Immobilization on Vascular and Lymphatic Endothelia
To activate integrins on rolling leukocytes, chemokines must be immobilized on HS GAGs of the endothelial glycocalyx (enlarged, inset). These GAGs can reduce, on the other hand, the strength of rolling adhesions. Chemokine transcytosis across the venule involves chemokine binding to subendothelial DARC or a related nonsignaling GPCR and to HS GAGs. Leukocyte sticking and crawling are followed by chemokine-triggered cell protrusions that sense HS GAG-immobilized chemokines within the ECM of the basement membrane.
The findings of Bao et al. (2010) further suggest that CCL21 presentation on lymphatic vessels is also HS GAG dependent and is necessary for optimal DC crossing of skin lymphatic vessels and subsequent entry into skin draining lymph nodes. Because substantial DC transport to lymph nodes remained intact in the absence of lymphatic HS GAGs, additional roles of soluble forms of other CCR7 ligands, secreted along the lymphatic tree remain possible (Bromley et al., 2008). This CCR7-dependent integrin-independent route is also taken by subsets of effector T cells, a process not addressed in this study.
Bao et al. (2010) also provide in vivo evidence for critical roles of endothelial HS in the immobilization and function of the inflammatory chemokine CXCL2 in neutrophil extravasation to inflamed skin. These results are consistent with previous in vitro evidence that lung capillary HS GAGs immobilize CXCL1 and CXCL2 and are critical for their transcytosis (Wang et al., 2005). The results also confirm a recent report on the role of HS GAGs in immobilization of exogenous CXCL2 on cremaster muscle venules in vivo (Massena et al., 2010). Notably, endothelial HS may also be necessary for chemokine exocytosis from Weibel Palade bodies, specialized granules that release CXCL8 and CCL26 in response to acute injury and allergic signals, a possibility that could be readily addressed in this Ext1-deficient model.
Importantly, Est1 inactivation in endothelial cells also abolished the decoration of functional HS moieties on endothelial secreted proteoglycans. Consequently, the basement membranes of both venules and lymphatic vessels tested was found to lack HS GAGs, whereas other stromal-derived GAGs such as lymph node fibroblastic reticular cells expressed intact HS and normally immobilized CCL21 (Bao et al., 2010). Intranodal migration of dendritic cells was therefore not affected, consistent with the dependence of this motility on stroma-presented and soluble CCR7 ligands.
In spite of these versatile functions of HS GAGs in different types of endothelial cells and basement membranes, it is still possible that many extravasation processes involve HS-independent mechanisms. For instance, several key lipid chemoattractants like PAF, leukotrienes, S1Ps, or 2-AG seem to immobilize directly on endothelial surfaces. Furthermore, as the density of endothelial HS is downregulated by HS-degrading enzymes during progressive inflammation, endothelial cells may directly secrete the chemokine they transcribe into tight synapses generated by adherent and transmigrating leukocytes.
Bao et al. (2010) report that in addition to serving as critical chemokine scaffolds, HS GAGs antagonize L-selectin-mediated rolling of lymphocytes on HEVs. HS GAGs were reported to decorate a nonfucosylated L-selectin ligand expressed by inflamed lung capillaries (Wang et al., 2005), yet this ligand and related HS ligands are either absent in HEVs or diluted out by the prototypic HEV L-selectin ligands, decorated with sulfated sLex. Furthermore, the HEV-expressed HS GAGs negatively regulate the adhesiveness of the prototypic sulfated sLex-decorated L-selectin ligands, probably via steric hindrance and electrostatic repulsion (Bao et al., 2010). It would be therefore interesting to test in the future whether and where HS-GAG-mediated electrostatic repulsion can downregulate additional leukocyte-endothelial interactions in endothelial beds other than HEVs.
The apparently preserved structure of Ext1-deleted blood and lymphatic vessels suggest that temporal deletion of HS does not impair basal signaling from critical endothelial growth factors known to bind HS GAGs. The endothelial conditional Ext1-deficient mice generated by Bao et al. (2010) will therefore be useful for future dissection of the roles of HS-chemokine interactions within other vascular beds, such as the bone marrow, thymus, spleen, gut, and lung. Similar approaches should be adopted to conditionally knock down HS biosynthesis in other chemokine-presenting cells such as epithelial cells, nerves, fibroblasts, pericytes, smooth muscle cells, and platelets ([Ley et al., 2007] and[Rot and von Andrian, 2004]). Furthermore, although HS GAGs on lymphocytes (Bao et al., 2010) and neutrophils (Wang et al., 2005) do not contribute to their inflammatory functions, subsets of DCs and macrophages may need to immobilize the chemokines they secrete within particular immune synapses. HS GAGs on subsets of leukemic cells may also promote autocrine chemokine activities essential for adhesion, motility, and survival. Interference with chemokine presentation on HS GAGs expressed by these various cellular systems could therefore be therapeutically promising for the manipulation of specific chemokine functions in different inflammatory and malignant processes.
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