Parisa Amjadi

Human BDCA-1-positive blood dendritic cells differentiate into phenotypically distinct immature and mature populations in the absence of exogenous maturational stimuli: differentiation failure in HIV infection.

Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b−/low and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b−/low population and absence on the CD11b+ cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b−/low DC but caused up-regulated expression in the CD11b+ cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.

Resting CD4+effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

BackgroundPreviously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.MethodsHere, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.ResultsAfter stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.ConclusionTaken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

Blockade of NKG2D ameliorates disease in mice with collagen-induced arthritis: a potential pathogenic role in chronic inflammatory arthritis.

OBJECTIVE To assess the role of the activating receptor NKG2D in arthritis. METHODS Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Resistance to regulatory T cell-mediated suppression in rheumatoid arthritis can be bypassed by ectopic foxp3 expression in pathogenic synovial T cells

Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.

Interleukin-10 regulates TNF-α−converting enzyme (TACE/ADAM-17) involving a TIMP-3 dependent and independent mechanism

IL‐10 is a potent anti‐inflammatory molecule, which regulates TNF‐α at multiple levels. We investigated whether IL‐10 also modulated the activity of the TNF‐α‐converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF‐α. In the presence of IL‐10, TNF‐α production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL‐10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase‐3 (TIMP‐3). We found that the inhibition of TACE activity at 2 h by IL‐10 was not TIMP‐3 dependent but that the late accumulation of surface TACE was prevented with TIMP‐3 antibodies. Furthermore, induction of endogenous TIMP‐3 was observed by western blotting in both LPS‐ and in LPS with IL‐10‐treated monocytes from 6 to 8 h of culture. These results indicate that IL‐10 further regulates TNF‐α by modulating TACE activation at early time points and by contributing to the induction of TIMP‐3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.

Activation of p38 mitogen-activated protein kinase is critical step for acquisition of effector function in cytokine-activated T cells, but acts as a negative regulator in T cells activated through the T-cell receptor

Peripheral blood CD4+ CD45RO+ T cells activated in vitro are able to induce expression of tumour necrosis factor‐α (TNF‐α) in monocytes via a contact‐dependent mechanism. Activation is achieved either with interleukin‐2 (IL‐2)/IL‐6/TNF‐α over an 8‐day period or cross‐linking CD3 using anti‐CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen‐activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti‐CD3 activated T cells, p38 MAPK is a negative regulator for anti‐CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte‐derived TNF‐α) or production of T‐cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine‐induced effector function and promotes cell proliferation and cytokine production.

Human blood CD1c dendritic cells stimulate IL‐12‐independent IFN‐γ responses and have a strikingly low inflammatory profile

Adaptive immune responses are initiated by resident myeloid tissue DC. A major fraction of tissue DC express CD1c+ and is thought to be derived from blood CD1c DC, an idea supported here by the observation that they express tissue‐homing molecules and rapidly differentiate into cells with a tissue DC phenotype. Responses are thought to be augmented/modulated further by inflammatory moDC. Although much accepted human myeloid DC cell biology is based on moDC studies, we find these 2 DC populations to be functionally distinct. Stimulated moDC produce high levels of IL‐10 and the Th1‐promoting cytokine IL‐12. Under identical conditions, CD1c DC synthesized no IL‐10 and no or low levels of IL‐12. Despite this, CD1c DC stimulated a strong Th1 response, demonstrated by IL‐12 neutralization to be IL‐12 independent, whereas the response induced by moDC was IL‐12 dependent. This finding was supported by studies on a patient with a highly reduced ability to synthesize IL‐12, whose CD1c DC induced a good Th1 response contrasting with the failure of his moDC, which were impaired in IL‐12 production, to induce IFN‐γ‐secreting T cells. The IL‐10 and IL‐12 data were confirmed by microarray analysis, which also showed that stimulated moDC produced inflammatory‐associated chemokines and cytokines, whereas stimulated CD1c DC showed minimal up‐regulation of these genes. Thus, moDC, widely used as a human myeloid DC model, do not faithfully reflect the properties of CD1c tissue DC, making the initial response to a pathogen or vaccine.