Paul Hutchinson

Macrophage migration inhibitory factor in rheumatoid arthritis: Evidence of proinflammatory function and regulation by glucocorticoids

OBJECTIVE Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor alpha (TNFalpha) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue. METHODS MIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1beta (IL-1beta), TNFalpha, or interferon-gamma (IFNgamma), and dexamethasone (DEX). Mononuclear cell TNFalpha release after exposure to FLS-conditioned medium was measured by ELISA. RESULTS MIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1beta, TNFalpha, or IFNgamma. In contrast, DEX 10(-7)M significantly reduced synoviocyte MIF, while DEX 10(-10)-10(-12)M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFalpha release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFalpha release. CONCLUSION These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.

Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

BACKGROUND Human endometrium is a highly regenerative tissue. We hypothesized that the source of endometrial stromal and vascular regeneration is a resident stromal stem/progenitor cell population. Putative human endometrial stromal stem/progenitor cells have been identified using clonal assays, a retrospective functional stem cell assay. Therefore, the aim of this study was to screen potential stem cell markers for the prospective isolation of human endometrial stromal stem/progenitor cells and to determine their capacity to identify colony-forming stromal cells. METHODS Single-cell suspensions of human endometrial stromal cells were sorted using fluorescence-activated cell sorting into positive and negative populations based on STRO-1, CD133, CD90 or CD146 expression for clonal assays. All markers were immunolocalized in human endometrium. RESULTS Small populations (2-9%) of human endometrial stromal cells expressed each of the markers. Only CD146(+) cells were enriched for colony-forming cells, and CD90(hi) cells showed a trend for greater enrichment compared with CD90(lo) cells. STRO-1 and CD146 were localized to perivascular cells of the endometrium. CD90 was strongly expressed by functionalis stroma and perivascular cells, but only weakly expressed in the basalis stroma. CD133 was expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. CONCLUSIONS This study identified CD146 as a marker of colony-forming human endometrial stromal cells supporting the concept that human endometrium contains a population of candidate stromal stem/progenitor cells.

Inhibitory effect of annexin I on synovial inflammation in rat adjuvant arthritis

OBJECTIVE Annexin I is an endogenous antiinflammatory mediator, expressed in rheumatoid arthritis (RA) synovium, the contribution of which to autoregulation of the synovial inflammatory response has not been examined in models of RA. We investigated the antiinflammatory role of annexin I in rat adjuvant arthritis. METHODS Rats with adjuvant-induced arthritis (AIA) were treated with a specific anti-annexin I monoclonal antibody (mAb), isotype control IgG, and/or dexamethasone. Clinical outcomes and synovial synthesis of tumor necrosis factor alpha (TNFalpha), prostaglandin E2 (PGE2), and nitric oxide were examined, and annexin I expression was assessed by flow cytometry and reverse transcription-polymerase chain reaction. RESULTS Anti-annexin I mAb reversed the effects of dexamethasone on the clinical features of AIA and exacerbated AIA in the absence of exogenous glucocorticoid. Clinical exacerbation of AIA by anti-annexin I mAb was accompanied by significantly increased synovial TNFalpha and PGE2, suggesting that annexin I tonically inhibits the production of these mediators. Anti-annexin I mAb treatment was associated with significantly reduced leukocyte intracellular annexin I, despite increased annexin I messenger RNA expression, consistent with a depletion effect of extracellular mAb via the cell surface. CONCLUSION Annexin I is a key endogenous inhibitory mediator of arthritis via mechanisms that include inhibition of cytokine and effector molecule production. Moreover, a synthesis-independent depletion of intracellular annexin I by extracellular antibody supports the hypothesis that externalization of annexin I is involved in its mode of action.

Functional integrity of granulosa cells from polycystic ovaries

OBJECTIVE The polycystic ovarian syndrome is frequently associated with human infertility and is a partially characterized syndrome of unknown aetiology. The aim of this study was to describe the functional integrity of granulosa cells from polycystic ovaries.

Anti-neutrophil monoclonal antibody therapy inhibits the development of adjuvant arthritis

The aim of this study was to determine the contribution of neutrophils to adjuvant arthritis (AA) by in vivo depletion of peripheral blood neutrophils. Specific anti‐neutrophil MoAb, RP3 (10 mg), or a control antibody was given twice daily on days 8–11 after injection of Mycobacterium tuberculosis in inbred male Sprague‐Dawley rats. RP3 treatment inhibited the neutrophil leukocytosis associated with AA (3.3 ± 0.6 × 103/mm3versus 21.2 ± 6.9 × 103/mm3; P < 0.001). On day 12, control animals exhibited severe arthritis as assessed by articular index (AI) (9.2 ± 1.3), increase in paw volume (149.3 ± 10.6%), and synovial fluid (SF) cell count (5.3 ± 0.5 × 105). RP3 treatment significantly reduced AI (1 ± 0.1; P < 0.001), paw volume (103.6 ± 5.8%; P < 0.001) and SF cells (0.6 ± 0.1 × 105; P < 0.001) without affecting cutaneous DTH (treated 0.6 ± 0.1 mm change in thickness, control 0.8 ± 0.2 mm; NS). Additional experiments demonstrated that CD4+ cell depletion but not decomplementation inhibited AA development and synovial neutrophil accumulation. Depletion of circulating neutrophils prevented joint inflammation and synovial leucocyte influx in AA, suggesting a pivotal role for neutrophils in the effector phase of AA. Inhibition of neutrophil accumulation by CD4+ cell depletion and not by decomplementation suggests that neutrophil accumulation in AA is T cell‐dependent.

Diagnosis of Mycobacterium tuberculosis infection using ESAT-6 and intracellular cytokine cytometry

Diagnosis of infection with Mycobacterium tuberculosis (MTB) using tuberculin skin testing (TST) is often hampered by prior Bacille Calmette–Guérin (BCG) vaccination. ESAT‐6 is a protein that is expressed by MTB but absent in BCG. It has been postulated that it might be useful in distinguishing MTB‐specific immune responses. This study measured CD4 T cell responder frequencies specific for ESAT‐6 and the TST reagent purified protein derivative (PPD) in patients with tuberculosis (n = 16), controls with non‐tuberculous pneumonia (n = 8) and normal subjects (n = 7). Responses were identified using the intracellular cytokine staining technique and flow cytometry on whole blood samples, and performed blinded to the patient condition. Antigen‐specific CD4 cells were defined by CD69 positivity and one or more cytokine [interleukin (IL)‐2, IL‐4, IL‐10, interferon (IFN)‐γ] and/or CD40L positivity. With ESAT‐6 stimulation it was found that TB patients had significantly higher frequencies of IFN‐γ and CD40L‐positive CD4 T cells compared to the normal group, while no significant differences were measured with PPD stimulation. A responder frequency of 0·01% or higher for at least one of the measured cytokines/CD40L was defined as a positive response. Using this criterion to compare the two patient groups, PPD had 100% sensitivity but 0% specificity while ESAT‐6 had 100% sensitivity and 88% specificity. Use of MTB‐specific proteins such as ESAT‐6 in combination with intracellular cytokine staining and flow cytometry has the potential to identify individuals with MTB infection.

Regulation of Annexin I in Rheumatoid Synovial Cells by Glucocorticoids and Interleukin-1

The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1β also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1β on cell surface annexin I. These results indicate that glucocorticoids and IL-1β upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.

Reduction in Arthritis Severity and Modulation of Immune Function in Tissue Factor Cytoplasmic Domain Mutant Mice

Tissue factor (TF), a transmembrane receptor for plasma factor VII(a), is the main initiator of the coagulation cascade. It has also been implicated in noncoagulant processes, including inflammation. The function of the TF cytoplasmic domain was studied in mice in which 18 of the 20 cytoplasmic amino acids were deleted. This mutation (TF(deltaCT/deltaCT)) is not associated with alterations in blood coagulation. Arthritis was induced by intra-articular injection of methylated bovine serum albumin (mBSA) in mice preimmunized with mBSA. Arthritis severity was significantly reduced in TF(deltaCT/deltaCT) mice compared to wild-type mice, including reductions in synovitis, synovial exudate, cartilage degradation, and bone damage. A marked reduction in synovial interleukin (IL)-1beta and IL-6 mRNA was also observed. Serum anti-mBSA IgG1, but not IgG2a, was increased in mutant mice. Cutaneous delayed-type hypersensitivity and antigen-induced T-cell proliferation were reduced in TF(deltaCT/deltaCT) compared to wild-type mice. A significant down-regulation of lipopolysaccharide-induced IL-1, tumor necrosis factor, IL-6, macrophage migration inhibitory factor, and matrix metalloproteinase-13 mRNA was observed in immunized, but not in naive TF(deltaCT/deltaCT) macrophages ex vivo. These data suggest a significant role for the cytoplasmic domain of TF in the regulation of the immunoinflammatory responses, a murine arthritis model, and macrophage function.

Glucocorticoid inhibition of adjuvant arthritis synovial macrophage nitric oxide production: role of lipocortin 1

Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid‐induced anti‐inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme‐digested synovium and treated with lipopolysaccharide (LPS) 1 μg/ml, dexamethasone (DEX) 10−7 M, and anti‐lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 ± 2.1 μmol/24 h). iNOS was detected in synovial macrophages (ED‐1+) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS‐induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti‐lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin 1 in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.

A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry.

Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens. Thirdly, microwave treatment also produces a discrete profile for DNA content analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.