Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

Characterization and recruitment of plasmacytoid dendritic cells in synovial fluid and tissue of patients with chronic inflammatory arthritis

Dendritic cells (DCs) are thought to play a key role in driving the immunopathogenic response underlying chronic inflammatory arthritis. In this study, we have examined the presence and phenotype of plasmacytoid DCs (pDCs) in the synovial fluids (SF) of patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), and osteoarthritis (OA) and determined the chemotactic properties of SF from these patients toward pDCs. Flow cytometry analysis showed that the percentage of pDCs, identified as a population of Lin(-)CD123(++) cells, is 4- to 5-fold higher in RA SF and PA SF than in OA SF. The morphological and immunophenotypic characterization of pDCs isolated from PA and RA SF indicates that they are in an immature state, most likely due to inhibitory factors present in RA SF, but are still able to undergo maturation when exposed ex vivo to viral agent or unmethylated DNA. CD123(+) and BDCA2(+) pDCs were detected by immunohistochemistry in RA synovial tissue in which expression of the IFN-alpha-inducible protein MxA was also found, suggesting production of type I IFN by maturing pDCs. We also show that CXCR3 and CXCR4 are expressed by both blood-derived pDCs and pDCs isolated from RA and PA SF and that CXCL-10, CXCL-11, and CXCL-12 present in RA and PA SF stimulate chemotaxis of blood-derived pDCs. Altogether, these findings suggest that chemokine-driven recruitment of pDCs from the blood to the inflamed synovium could be important in the regulation of the immune response in chronic inflammatory arthritis.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

Introduction

The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.

Methods

The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.

Results

Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68⁺ and CD163⁺ macrophages, CD55⁺ fibroblast-like synoviocytes (FLS), CD31⁺ endothelial cells or infiltrating monocytes and CD19⁺ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.

Conclusions

The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.     

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane,synovial fluid and peripheral blood

Introduction

CD4⁺CD25^+/highCD127^low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.

Methods

Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.

Results

CD4⁺ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO⁺RA^-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.

Conclusions

Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L^-CD69⁺), whereas peripheral Tregs are resting central memory cells (CD62L⁺CD69^-).     

Impairment in the telocyte/CD34+ stromal cell network in human rheumatoid arthritis synovium

Telocytes (TCs)/CD34⁺ stromal cells have recently emerged as peculiar interstitial cells detectable in a variety of organs throughout the human body. TCs are typically arranged in networks establishing unique spatial relationships with neighbour cells and likely contributing to the maintenance of tissue homeostasis by both cell-to-cell contacts and releasing extracellular vesicles. Hence, TC defects are being increasingly reported in different pathologies, such as chronic inflammatory and fibrotic conditions. In this regard, TCs/CD34⁺ stromal cells have been shown to constitute an intricate interstitial network in the subintimal area of the normal human synovial membrane, but whether they are altered in chronic synovitis has yet to be explored. We therefore undertook a morphologic study to compare the distribution of TCs/CD34⁺ stromal cells between normal synovium and chronically inflamed synovium from patients with rheumatoid arthritis (RA) by using CD34 immunohistochemistry and CD31/CD34 double immunofluorescence. CD34 immunostaining revealed that, at variance with normal synovium, the inflamed and hyperplastic RA synovial tissue was nearly or even completely devoid of TCs/CD34⁺ stromal cells. Double immunofluorescence confirmed that almost all CD34⁺ tissue components detectable in RA synovium were blood vessels coexpressing CD31, while a widespread network of CD31⁻/CD34⁺ TCs was clearly evident in the whole sublining layer of normal synovium. In the context of the emerging diverse roles of TCs/CD34⁺ stromal cells in the regulation of tissue homeostasis and structure, the remarkable impairment in their networks herein uncovered in RA synovium may suggest important pathophysiologic implications that will be worth investigating further.     

Structural and Immunological Effects of Skin Cryoablation in a Mouse Model

Cryoablation is therapeutically applied for various disorders in several organs, and skin diseases are typical targets as this cryotherapy has been widely used for viral warts, benign tumors, and actinic keratosis. The main mechanisms of cryoablation consist of direct freezing effect on skin constituents, thrombosis formation in microcirculation, and subsequent immunological responses. Among them, however, the immunological mechanism remains unelucidated, and it is an issue how the direct freezing injury induces immunological consequences. We established a mouse cryoablation model with liquid nitrogen applied to the shaved back skin, and used this system to study the immunological excitement. After application of liquid nitrogen, the thermal decrease ratio was -25°C/sec or less and the lowest temperature was less than -100°C, which was sufficient to induce ulceration. Destruction of cornified layer and necrosis of epidermal cells were observed in transmission electron microscopy image, and increased transepidermal water loss and skin permeability were detected by the functional measurements. By flow cytometry, antigen-presenting dendritic cells (DCs), including PDCA1⁺B220⁺CD19^- plasmacytoid DCs (pDCs) and CD11c⁺ myeloid DCs, as well as neutrophils and macrophages were increased in subcutaneous tissue. In parallel, the mRNA expressions of interferon α1 which are known as pDC-producing cytokines, was elevated. We also found marked degranulation of mast cells, providing a possibility that released histamine attracts pDCs. Finally, FITC migration assay revealed that pDCs and CD11c⁺ DCs emigrated from the cryoablated skin to the draining lymph nodes. Our study suggests that cryoablation induces destruction of the barrier/epidermis, accumulation of pDCs and CD11c⁺ DCs to the skin, and migration of DCs to regional lymph nodes. Viral elements or tumor cell lysates released from damaged keratinocytes may stimulate the DCs, thereby leading to antiviral or antitumor effect.     

IL-9, a local growth factor for synovial T cells in inflammatory arthritis

ObjectiveThe regulatory role of the T_h9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis.MethodsCD3⁺ T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3⁺ T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9.ResultsSF of PsA and RA was enriched with IL-9 producing CD3⁺ T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3⁺ T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p < .005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway.ConclusionIL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.     

Dendritic cells (DCs) in rheumatoid arthritis (RA): progenitor cells and soluble factors contained in RA synovial fluid yield a subset of myeloid DCs that preferentially activate Th1 inflammatory-type responses

There is evidence that mature dendritic cells (DCs) present in the rheumatoid arthritis (RA) joint mediate immunopathology in RA. In this study, we indicate that early myeloid progenitors for DCs and DC growth factors existing in RA synovial fluid (SF) are also likely participants in the RA disease process. A fraction of cells lacking markers associated with mature DCs or DC precursors and enriched in CD34(negative) myeloid progenitors was isolated from RA SF. These cells proliferated extensively when cultured in vitro with cytokines that promote the growth of myeloid DCs (GM-CSF/TNF/stem cell factor/IL-4) and, to a lesser degree, when cultured with monocyte/granulocyte-restricted growth factors (M-CSF/GM-CSF). Mature DCs derived from RA SF progenitors with CD14-DC cytokines known to be prevalent in the inflamed RA joint (GM-CSF/TNF/stem cell factor/IL-13) were potent stimulators of allogeneic T cells and inflammatory-type Th1 responses and included CD14-DC subtypes. Cell-free RA SF facilitated DC maturation from myeloid progenitors, providing direct evidence that the inflamed RA joint environment instructs DC growth. Enhanced development of CD14-derived DCs was correlated with the presence of soluble TNFR (p55), raising the possibility that soluble TNFR also regulate CD14-derived DC growth in vivo. SF from patients with osteoarthritis contained neither myeloid DC progenitors nor DC growth factors. The existence of DC progenitors and myeloid DC growth factors in RA SF supports the concept that RA SF may be a reservoir for joint-associated DCs and reveals a compelling mechanism for the amplification and perpetuation of DC-driven responses in the RA joint, including inflammatory-type Th1 responses.     

Similar functional activity of dendritic cells recruited to the mesenteric lymph nodes of newborn and adult mice after the rectal delivery of Mycobacterium bovis BCG

BCG rectal administration to newborn and adult mice induced protective immune responses against tuberculosis. BCG reaches the sub-epithelial site and the draining mesenteric lymph nodes (MLNs), and dendritic cells (DC) could be recruited to these sites. Using polarized Caco-2 epithelial cells, we showed that BCG translocates epithelial cells to basolateral compartment. Delayed in newborn BALB/c mice, an important recruitment of CD11c⁺ DCs, was documented in the rectal lamina propria and the MLNs during the first two weeks after rectal BCG delivery. In MLNs, two major DC subtypes were observed: conventional DCs (cDCs) (B220⁻) and plasmacytoid DCs (pDCs) (B220⁺). CIRE, mouse DC-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is predominantly expressed on pDCs and at a higher level on pDCs from the adult compared to newborn MLNs. cDCs with a higher capacity to induce the proliferation of naïve CD4⁺ T cells than pDCs, triggered CD4⁺ T cells to produce interferon-γ whereas pDCs triggered them to release interleukin-10. Both DC subtypes equilibrates T cells as a source of microbicidal/microbiostatic signals and those acting as source of counter-inflammatory signals, preventing tissue damage and/or accelerating tissue repair. Thus, rectal delivery of BCG could be a safe and efficient route of vaccination against tuberculosis.     

CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR^-CD14⁺ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR^-CD14⁺ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d^? cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.     

Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.     

The in vitro effect of methylprednisolone on the processes of activation of CD4<Superscript>+</Superscript>CD45RO<Superscript>+</Superscript> T-cells from healthy subjects and rheumatoid-arthritis patients

Flow cytometry has been used to analyze the changes in the number of CD4⁺ cells that expressed surface markers of activation (CD25, CD71, HLA-DR, and CD95) in cultures of TCR-stimulated CD3⁺CD45RO⁺ Т-lymphocytes after in vivo exposure to different concentrations of methylprednisolone (MP). T-cells were obtained from healthy donors and rheumatoid arthritis (RA) patients. Suppressive action of МР on the expression of activation and proliferation markers (CD25 and CD71, respectively) by CD4⁺ T-cells was observed in all study subjects. МР increased the number of CD4⁺ HLA-DR⁺/CD95⁺ cells among the СD3⁺CD45RO⁺ cells obtained from RA patients and subjected to TCR activation, whereas the number of such cells in the control group decreased after MP treatment. The MP-induced changes in the cells subjected to TCR activation can be indicative of relative resistance of the CD4⁺CD45RO⁺HLA-DR⁺/CD95⁺ cell population in RA patients to the action of glucocorticoids and the possible role of this subpopulation in RA pathogenesis.     

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis.

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.     

Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effects of IL-10.

IL-10 down-regulates the APC function of many dendritic cells (DC), including human peripheral blood (PB) DC. In rheumatoid arthritis (RA), synovial fluid (SF) DC express markers of differentiation and are effective APC despite abundant synovial IL-10. The regulation of DC responsiveness to IL-10 was therefore examined by comparing the effect of IL-10 on normal PB and RA SF DC. Whereas IL-10 down-modulated APC function and MHC class II and B7 expression of PB DC, IL-10 had no such effect on SF DC. Since SF DC have differentiated in vivo in the presence of proinflammatory cytokines, PB DC were cocultured in the presence of IL-10 and either GM-CSF, IL-1beta, TNF-alpha, IL-6, or TGF-beta. GM-CSF, IL-1beta, and TNF-alpha were all able to restore APC function. Whereas the effects of IL-10 on PB DC were shown to be mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1 protein was demonstrated in PB and SF DC whole cell lysates, suggestive of predominant intracellular localization of the receptor. Thus, DC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R1 expression or signaling.     

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Introduction

TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.

Methods

TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.

Results

TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38⁺ plasma cells and with CD22⁺ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22⁺ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1⁺ osteoblasts.

Conclusions

The expression of TWEAK by CD22⁺ B cells and CD38⁺ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.