Mike Salmon

Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation

Fibroblasts are important sentinel cells in the immune system and, here, it is proposed that these cells play a critical role in the switch from acute inflammation to adaptive immunity and tissue repair. It is suggested that chronic inflammation occurs because of disordered fibroblast behaviour in which failure to switch off their inflammatory programme leads to the inappropriate survival and retention of leukocytes within inflamed tissue.

Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis‐prone population

Anergic/suppressive CD4+CD25+ T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4+CD25+ T cell subset, which constitutes 7–10 % of CD4+ T cells in peripheral blood and tonsil. These cells are a CD45RO+CD45RBlow highly differentiated primedT cell population that is anergic to stimulation. Depletion of this small subset from CD4+ T cells significantly enhances proliferation by threefold in the remaining CD4+CD25– T cells, while the addition of isolated CD4+CD25+ T cells to CD4+CD25– T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL‐4, IL‐10 or TGF‐β and is cell‐contact dependent. Isolated CD4+CD25+ T cells are susceptible to apoptosis that is associated with low Bcl‐2 expression, but this death can be prevented by IL‐2 or fibroblast‐secreted IFN‐β. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis‐prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.

RGD peptides induce apoptosis by direct caspase-3 activation

Synthetic peptides containing the arginine–glycine–aspartate (RGD) motif have been used extensively as inhibitors of integrin–ligand interactions in studies of cell adhesion, migration, growth and differentiation,,, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of pro-caspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate–aspartate–methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD–DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent pro-apoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis,,.

Persistent induction of the chemokine receptor CXCR4 by TGF-beta 1 on synovial T cells contributes to their accumulation within the rheumatoid synovium.

Chemokines and their receptors determine the distribution of leukocytes within tissues in health and disease. We have studied the role of the constitutive chemokine receptor CXCR4 and its ligand, stromal-derived factor-1 (SDF-1) in the perivascular accumulation of T cells in rheumatoid arthritis. We show that synovial T cells, which are primed CD45RO+CD45RBdull cells and consequently not expected to express constitutive chemokine receptors, have high levels of the chemokine receptor CXCR4. Sustained expression of CXCR4 was maintained on synovial T cells by specific factors present within the synovial microenvironment. Extensive screening revealed that TGF-β isoforms induce the expression of CXCR4 on CD4 T cells in vitro. Depletion studies using synovial fluid confirmed an important role for TGF-β1 in the induction of CXCR4 expression in vivo. The only known ligand for CXCR4 is SDF-1. We found SDF-1 on synovial endothelial cells and showed that SDF-1 was able to induce strong integrin-mediated adhesion of synovial fluid T cells to fibronectin and ICAM-1, confirming that CXCR4 expressed on synovial T cells was functional. These results suggest that the persistent induction of CXCR4 on synovial T cells by TGF-β1 leads to their active, SDF-1-mediated retention in a perivascular distribution within the rheumatoid synovium.

Inhibition of T cell apoptosis in the rheumatoid synovium.

Synovial T cells in rheumatoid arthritis are highly differentiated and express a phenotype suggesting susceptibility to apoptosis (CD45RB dull, CD45RO bright, Bcl-2 low, Bax high, Fas high). However, no evidence of T cell apoptosis was found in synovial fluid from any of 28 patients studied. In contrast, synovial fluid from 10 patients with crystal arthritis showed substantial levels of T cell apoptosis. The failre of apoptosis was not an intrinsic property of rheumatoid synovial T cells, as they showed rapid spontaneous apoptosis on removal from the joint. Synovial T cells from rheumatoid arthritis and gout patients could be rescued from spontaneous apoptosis in vitro either by IL-2R gamma chain signaling cytokines (which upregulate Bcl-2 and Bcl-XL) or by interaction with synovial fibroblasts (which upregulates Bcl-xL but not Bcl-2). The phenotype of rheumatoid synovial T cells ex vivo (Bcl-2 low, Bcl-xL high) suggested a fibroblast-mediated mechanism in vivo. This was confirmed by in vitro culture of synovial T cells with fibroblasts which maintained the Bcl-xL high Bcl-2 low phenotype. Synovial T cells from gout patients were Bcl-2 low Bcl-xL low and showed clear evidence of apoptosis in vivo. Inhibition experiments suggested that an integrin-ligand interaction incorporating the Arg-Gly-Asp motif is involved in fibroblast-mediated synovial T cell survival. We propose that environmental blockade of cell death resulting from interaction with stromal cells is a major factor in the persistent T cell infiltration of chronically inflamed rheumatoid synovium.

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vβ repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RBlow subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen‐specific stimulation in vivo. This suggests that anergic/suppressiveCD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non‐professional antigen‐presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.

Cytomegalovirus-Specific CD4+ T Cells in Healthy Carriers Are Continuously Driven to Replicative Exhaustion

Repeated antigenic encounter drives proliferation and differentiation of memory T cell pools. An important question is whether certain specific T cells may be driven eventually to exhaustion in elderly individuals since the human life expectancy is increasing. We found that CMV-specific CD4+ T cells were significantly expanded in healthy young and old carriers compared with purified protein derivative-, varicella zoster virus-, EBV-, and HSV-specific populations. These CMV-specific CD4+ T cells exhibited a late differentiated phenotype since they were largely CD27 and CD28 negative and had shorter telomeres. Interestingly, in elderly CMV-seropositive subjects, CD4+ T cells of different specificities were significantly more differentiated than the same cells in CMV-seronegative individuals. This suggested the involvement of bystander-secreted, differentiation-inducing factors during CMV infection. One candidate was IFN-α, which induced loss of costimulatory receptors and inhibited telomerase in activated CD4+ T cells and was secreted at high levels by CMV-stimulated plasmacytoid dendritic cells (PDC). The CMV-specific CD4+ T cells in elderly subjects had severely restricted replicative capacity. This is the first description of a human memory T cell population that is susceptible to being lost through end-stage differentiation due to the combined effects of lifelong virus reactivation in the presence of bystander differentiation-inducing factors.

Inhibition of fibrocyte differentiation by serum amyloid P

Wound healing and the dysregulated events leading to fibrosis both involve the proliferation and differentiation of fibroblasts and the deposition of extracellular matrix. Whether these fibroblasts are locally derived or from a circulating precursor population is unclear. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that enter sites of tissue injury to promote angiogenesis and wound healing. We have found that CD14+ peripheral blood monocytes cultured in the absence of serum or plasma differentiate into fibrocytes within 72 h. We purified the factor in serum and plasma that prevents the rapid appearance of fibrocytes, and identified it as serum amyloid P (SAP). Purified SAP inhibits fibrocyte differentiation at levels similar to those found in plasma, while depleting SAP reduces the ability of plasma to inhibit fibrocyte differentiation. Compared with sera from healthy individuals and patients with rheumatoid arthritis, sera from patients with scleroderma and mixed connective tissue disease, two systemic fibrotic diseases, were less able to inhibit fibrocyte differentiation in vitro and had correspondingly lower serum levels of SAP. These results suggest that low levels of SAP may thus augment pathological processes leading to fibrosis. These data also suggest mechanisms to inhibit fibrosis in chronic inflammatory conditions, or conversely to promote wound healing.

Cellular environments and apoptosis: tissue microenvironments control activated T-cell death.

Activated T cells must be removed by apoptosis at the end of an immune response in order to maintain cellular homeostasis. Although recent attention has focused on the role of CD95 (Fas/APO-1) in the elimination of activated T cells, apoptosis can also be induced by cytokine deprivation. Here, Arne Akbar and Mike Salmon describe how both death pathways interact in activated T cells and are profoundly influenced by different tissue microenvironments.

Ectopic expression of the B cell-attracting chemokine BCA-1 (CXCL13) on endothelial cells and within lymphoid follicles contributes to the establishment of germinal center-like structures in Sjogren's syndrome

OBJECTIVE To test the hypothesis that the formation of ectopic germinal center (GC)-like structures in Sjögrens syndrome (SS) is associated with the ectopic expression of the constitutive lymphoid tissue-homing chemokines B cell-attracting chemokine 1 (BCA-1; or, CXCL13) and stromal cell-derived factor 1 (SDF-1; or, CXCL12). METHODS Immunohistochemical and immunofluorescence analysis was used to determine the expression of the constitutive chemokines BCA-1 (CXCL13) and SDF-1 (CXCL12) in salivary glands from 5 SS patients and 3 non-SS patients. In addition, the expression of their respective receptors (CXCR5 and CXCR4) was examined on infiltrating lymphocytes. Human tonsil was used as a positive control for secondary lymphoid tissue. RESULTS BCA-1 (CXCL13) was expressed within lymphoid aggregates in SS, which shared many structural features with GCs in tonsil. BCA-1 (CXCL13) was completely absent in control biopsy samples from patients who did not have SS. High levels of BCA-1 (CXCL13) were also found on endothelial cells in salivary glands from SS patients. Diseased SS tissue was infiltrated by CXCR5-expressing B cells which organized into GC-like clusters. In complete contrast, SDF-1 (CXCL12), a constitutive chemokine involved in leukocyte retention within lymphoid tissue, was expressed by epithelial cells in both diseased and control samples. The chemokine receptor for SDF-1, CXCR4, was expressed on T cells that accumulated in a periductal distribution in diseased tissue. CONCLUSION The ectopic expression of BCA-1 (CXCL13) on endothelial cells and within GC-like structures, together with the strong expression of SDF-1 (CXCL12) on ductal epithelial cells, is a unique feature of inflamed glands in SS. By creating a local microenvironment supportive of focal B cell aggregation and differentiation, with structural features that are remarkably similar to GCs, BCA-1 (CXCL13) and SDF-1 (CXCL12) may contribute to the excessive production of high-affinity, class-switched autoantibodies and to the high incidence of B cell lymphomas classically associated with SS.