Selective sequestration of inflammatory chemokines is critical for the successful resolution of inflammatory responses in vivo. D6 is an atypical chemokine receptor that scavenges inflammatory chemokines and is pivotal in resolving models of chemokine-driven cutaneous inflammation. We provide evidence that expression of D6 is not limited to the lymphatic endothelium at sites of inflammation as previously believed. Instead we postulate that D6 expression in leucocytes may have a significant impact upon chemokine bioavailability during the resolution phase of inflammation. D6 expressed on the lymphatic endothelia may instead have complementary roles in preventing inappropriate leucocyte migration to the lymph node by keeping the endothelium free from inflammatory chemokines.

The group of chemotactic cytokines, otherwise known as chemokines, are an important class of attractant molecules that mediate the migration of leucocytes into and out of tissues. Chemokines are instrumental in orchestrating the instigation and maintenance of inflammatory responses. Inflammatory mediators, such as tumour necrosis factor and microbial products, are well-characterized innate inducers of chemokines that initiate the influx of leucocytes into inflamed areas. Incoming leucocytes can then participate in fighting infection, contribute to the removal of tissue debris and assist in tissue repair [13]. In most cases, once an inflamed or damaged tissue has been repaired and pathogens eliminated, inflammation subsides and the tissue returns to its homoeostatic norm. In some individuals, the resolution phase of chemokine-driven inflammation is ineffective and a number of chronic inflammatory diseases may be perpetuated by the ongoing production of chemokines [46]. Studies have begun to highlight how the removal of these potent inflammatory agents are critical for the resolution of immune responses and are well reviewed on pp. 1009–1013 of this issue by Hansell et al. [6a]. There are likely to be a number of mechanisms by which chemokines are regulated in the extracellular space of inflamed tissues, such as tissue-resident proteases and binding by GAGs (glycosaminoglycans) [7]. However, recent studies have highlighted the pivotal role that chemokine scavenging by non-signalling chemokine receptors have in resolving models of cutaneous inflammation [8,9]. The atypical chemokine receptor D6 is a non-signalling receptor that binds a broad range of inflammatory CC chemokines with high affinity. In vitro results indicate that chemokine bound to D6 is internalized following which it dissociates from D6 in acidic endosomes. Dissociated chemokine is then degraded, while D6 is recycled to the surface, so that one D6 molecule is able to scavenge multiple chemokine molecules [1014]. Mice lacking D6 demonstrate excessive levels of chemokine for a prolonged period after application of the irritant PMA to the skin. This precipitates the development of a sustained and damaging inflammatory response that is reminiscent of psoriasis in humans [8]. Independently, another study has shown that D6-deficient mice develop exaggerated responses to complete Freund's adjuvant and exhibit increased levels of CC chemokine and cellularity in draining lymph nodes [9].

These reports lend much support to the notion that D6 is important for resolving inflammatory responses in vivo. This model has, to date, been argued from the perspective that D6 is only expressed on lymphatic endothelium [15], principally because the initial immunohistochemical analysis of D6 expression in non-inflamed human tissue identified lymphatic endothelium as the primary site of expression [16]. However, the expression of D6 within inflamed tissues was not reported. It remains possible that other D6-expressing cell types may be important in the inflamed context. While the demonstration of human D6 expression by lymphatic endothelium has led to the postulation that these cells play a principal role in this context, much of the literature on lymphatic vasculature suggests that it may not have sufficient density or spread to function as a tissue-wide regulator of CC chemokine levels [1719]. This suggests that LECs (lymphatic endothelial cells) may not be the primary regulators of chemokine scavenging in inflamed tissues and that other D6-expressing cell types may contribute more effectively to the resolution of inflammation.

We have begun studying the promoter for human and mouse D6. Initial in silico analysis of the upstream sequence has reveal a multitude of binding sites for transcription factors that are associated with cells of the haemopoietic lineage. The putative promoter of both mouse and human D6 contain consensus binding sites for a range of transcription factors involved in haemopoietic cell differentiation such as Ikaros, Myb and GATA1. This is suggestive of transcriptionally regulated expression in T-cells and DCs (dendritic cells) (Ikaros), megakaryocytes (Myb) and stem cells, mast cells, eosinophils and megakaryocytes (GATA). Work is ongoing to investigate the functional significance of these transcription factor-binding sites.

Interestingly, while the earlier immunohistochemical studies did not examine D6 expression in inflamed tissues, more recent analysis specifically in inflamed human tissues has identified, in addition to the lymphatic endothelium, large numbers of leucocyte-like cells as being immunopositive for D6 (C.S. McKimmie, M. Pruenster, A. Rot and G. Graham, unpublished work). This is consistent with our previous demonstration of D6 transcripts in murine and human leucocyte populations, raising the possibility that leucocyte-expressed D6 could contribute significantly to its in vivo functions [10,11]. Preliminary work using quantitative PCR has highlighted the expression in a wide range of leucocytes in both human and mouse cells (see Table 1) and has shown that expression is inducible (C.S. McKimmie, M. Pruenster, A. Rot and G. Graham, unpublished work). Given the widespread distribution of leucocytes and their ability to migrate directly to sites of inflammation, we hypothesize that leucocyte D6 may be the primary cellular vehicle for D6-dependent chemokine degradation and regulation of inflammation.

Table 1
Absolute expression of D6 (normalized to the housekeeping genes, as described previously [24]) was determined by quantitative PCR using cDNA from resting isolated leucocyte subsets and leucocyte cell lines

This Table summarizes results from our laboratory (C.S. McKimmie, M. Pruenster, A. Rot and G. Graham, unpublished work).

Cell type Expression 
Human  
 BeWo (choriocarcinoma) High 
 K562 (haemopoietic progenitor) Medium 
 Primary macrophages Medium 
 THP-1 (monocytic cell line) Medium 
 Mast cells Medium 
 Jurkat (T-cell line) Low 
 Molt-4 (T-cell line) Low 
 Primary monocytes Low 
Mouse  
 Mast cells Medium 
 B-cells Medium/low 
 Dendritic cells Medium/low 
 CD4+ T-cells Low 
 Macrophages Low 
 Neutrophils Low 
Cell type Expression 
Human  
 BeWo (choriocarcinoma) High 
 K562 (haemopoietic progenitor) Medium 
 Primary macrophages Medium 
 THP-1 (monocytic cell line) Medium 
 Mast cells Medium 
 Jurkat (T-cell line) Low 
 Molt-4 (T-cell line) Low 
 Primary monocytes Low 
Mouse  
 Mast cells Medium 
 B-cells Medium/low 
 Dendritic cells Medium/low 
 CD4+ T-cells Low 
 Macrophages Low 
 Neutrophils Low 

Together, this suggests that lymphatic endothelial D6 may play a distinct role in the context of lymphatic endothelial and lymph node cell biology [19]. Immature DCs migrate to inflamed and infected peripheral tissues under the control of inflammatory chemokines, while migration of matured DCs to the draining lymph nodes for antigen presentation is dependent upon induction of CCR7 (CC chemokine receptor 7) [20]. The same is true of T-cell entry and exit from peripheral tissues [21]. The egress of these cells from the tissues is aided by the expression of the CCR7 ligand, CCL21 (CC chemokine ligand 21), by lymphatic endothelium [22]. We now propose that the specific role for lymphatic endothelial D6 is to denude the surface and vicinity of the endothelium of inflammatory CC chemokines so that available GAGs present only homoeostatic chemokines, primarily CCL21 [19]. This would ensure that DCs will only leave the peripheral tissues upon up-regulation of CCR7 and will not interact with lymphatic endothelium through immobilized inflammatory CC chemokines. The same model is likely to hold with T-cells. In D6-null mice, we propose that the absence of D6 will mean that inflammatory CC chemokines will be displayed, along with CCL21, on the surface of the lymphatic endothelium and will allow the inappropriate interaction of inflammatory CC chemokine receptor-bearing cells, such as immature DCs, with the endothelium. This may trigger the egress of inflammatory CCR-bearing, but CCR7-negative, cells from inflamed sites. Furthermore, due to the high level of inflammatory chemokine production at sites of inflammation, it is likely that, without lymphatic D6, they will out-compete CCL21 for binding on the lymphatic endothelium, preventing CCR7/CCL21-mediated migration altogether, leading paradoxically to the retention of matured APCs (antigen-presenting cells) in the tissues and inappropriate early egress of immature inflammatory CC chemokine receptor-bearing cells. The premature egress of immature DCs (and of T-cells) from peripheral inflamed or infected tissues would be likely to interfere with the proper initiation of the immune response within the draining lymph nodes. Intriguingly, and in agreement with this model, recent results have shown that D6-null mice display a deficient immune response in auto-immune models of multiple sclerosis [23]. Critically, these mice demonstrate an otherwise inexplicable retention of DCs within inflamed tissue, which is probably accounted for by the proposed deregulated migration of matured DCs to the draining lymph nodes.

We provide findings that suggest leucocytes may play a significant role in chemokine scavenging through D6-mediated sequestration. We present preliminary results that suggest D6 expression in a range of leucocytes in vitro and D6 expression in leucocytes within inflamed human tissue. In addition, initial analysis of the D6 promoter has revealed a multitude of binding sites for haemopoietic-associated transcription factors. The expression of D6 on leucocytes may require a modification of our understanding of in vivo D6 function and suggests that lymphatic endothelial D6 may perform additional and distinct roles through the sequestration of inflammatory chemokines.

Control of Immune Responses: A Focus Topic at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by B. Foxwell (Imperial College London, U.K.), G. Graham (Glasgow, U.K.), R. Nibbs (Glasgow, U.K.) and S. Ward (Bath, U.K.).

Abbreviations

     
  • CCL21

    CC chemokine ligand 21

  •  
  • CCR

    CC chemokine receptor

  •  
  • DC

    dendritic cell

  •  
  • GAG

    glycosaminoglycan

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