R J Looney

CD40 Expression by human fibroblasts

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.

Allergic reactions to cyclophosphamide: Delayed clinical expression associated with positive immediate skin tests to drug metabolites in five patients

BACKGROUND Cyclophosphamide (CP) is one of the relatively few drugs implicated in systemic allergic reactions for which the metabolites are well known. Formation of CP metabolites is a multistep, time-dependent process (hours) with significant interindividual differences. Although allergic reactions to CP have been recorded in 17 previous reports, skin testing with CP or its metabolites has been included in only five. We now describe five patients receiving monthly cycles of intravenous CP whose allergic reactions included clinical features of type I hypersensitivity but were atypical in their markedly delayed onset (i.e., 8 to 16 hours in patients 1 to 4 and 10 days in patient 5). OBJECTIVE The objective was to investigate these late-developing clinical reactions by skin testing with CP and two of its major metabolites. METHODS The five patients and a control group receiving intravenous CP uneventfully were studied by the same skin test protocol. RESULTS The four individuals in the control group were unreactive to CP or its metabolites. All five patients with late-onset allergic reactions had positive immediate skin test results to CP metabolites but not to CP itself. We propose that the allergic reactions in patients 1 to 4 were mediated, wholly or in major part, by IgE antibodies reactive with allergens derived from time-dependent drug metabolites. The 10-day lag time in patient 5 is unexplained. Immunomodulation by the underlying malignancies or by the immunosuppressive drugs could have contributed. CONCLUSION IgE-mediated allergic drug reactions may have a delayed onset if the allergen is a time-dependent drug metabolite, illustrated in this study by CP.

ACCUMULATION OF INTERFERON GAMMA-PRODUCING TH1 HELPER T CELLS IN NASAL POLYPS

We investigated the mechanisms involved in the formation of nasal polyps by examining T-cell clones and their production of soluble mediators in nasal polyps. Recently, the allergic origin of nasal polyps has been challenged. To study this question we characterized T cells from polyp tissue of allergic individuals in terms of their cytokine pattern. Nasal polyp T cells were cloned from allergic individuals undergoing polypectomy. Polyp tissue was dispersed enzymatically, and T cells were stimulated with mitogen and interleukin-2. Control T cells were obtained from peripheral blood of nonallergic donors. Cytokine production of interleukin-4 and interferon was then determined by indirect enzyme-linked immunosorbent assay tests. Polyp T-cell clones were found to produce high interferon but low interleukin-4 levels that were not significantly different from control peripheral blood T-cell clones. In addition, immunoglobulin production by dispersed polyp tissue was investigated. Immunoglobulin levels were higher in polyp tissues than in serum with immunoglobulin A predominating. These results suggest that the inflammatory reaction in nasal polyps is different than that seen in a typical type I hypersensitivity response.

PS7:130 Interferon beta blockade rescues human bm-msc osteoblastogenesis defects in systemic lupus erythematosus

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent stem cells that can differentiate into chondrocytes, osteoblasts and adipocytes. SLE has been implicated as a stem cell disorder with impaired immunomodulatory function of SLE BM-MSCs and improvement of lupus nephritis with healthy MSCs transplantation has been suggested. However, the exact differentiation defects of SLE BM-MSCs have not been addressed, nor and potential interventions studied. Our previous work indicates upregulation of IFN beta specific genes in human SLE bone marrow derived MSCs compared to normal bone marrow MSC. Here we set out to investigate the differentiation defects of SLE BM-MSCs and potential intervention approaches. We compared 6 age paired BM aspirates from healthy controls and SLE patients. BM-MSCs from SLE patients and healthy controls were isolated and cultured. The MSC surface markers are positive for CD73, CD90 and CD105, but negative for CD34 and CD45 in both healthy and SLE BM-MSCs after culture. No difference was observed in the surface markers between SLE and healthy BM-MSCs. However, SLE MSCs display significantly reduced osteoblastogenesis markers, such ALP (6 fold, p<0.05), RUNX2 (8 fold, p<0.05), OCN (4 fold, p<0.05) and BSP (4 fold, p<0.05). The osteoblast induction and ALP staining analysis for osteoblastogenesis also suggested a reduced differentiation with the SLE BM-MSCs. In contrast to the downregulation of osteoblast markers, the expression of IFN beta is increased 5 fold (p<0.05) in SLE BM-MSCs. When BM-MSCs from healthy controls were treated with IFN beta for 6 hours, reduced ALP (12 fold, p<0.05), RUNX2 (11 fold, p<0.05), OCN (8 fold, p<0.05) and BSP (7 fold, p<0.05) were observed, suggesting that IFN beta plays an important role in inhibiting SLE BM-MSC differentiation into osteoblasts. Conversely, when IFN beta neutralising antibody was applied to SLE BM-MSCs, the osteoblastogenesis markers were significantly enhanced. IFN-I signature is an important feature of SLE. Our present work suggests that SLE BM-MSCs produce IFN beta, mediating a decrease in osteoblastogenesis capacity. The successful rescue of the SLE BM-MSCs osteoblastogenesis defect with an IFN beta neutralising antibody highlights IFN as a new potential therapeutic target for SLE treatment.

I-02 Increased interferon β expression and senescence associate secretory phenotype impair the immunomodulatory function of bone marrow mesenchymal stromal cells in patients with systemic lupus erythematosus

Background Interferon I (IFN-I) signature is an important feature of systemic lupus erythematosus (SLE). Our previous study identified an IFN-I signature in both bone marrow (BM) and peripheral blood of SLE patients. The overlapping roles of IFNα subtypes and disappointing results with IFNα subtype blockade in clinical trials calls for alternative targets and recent findings centred on IFNβ suggest that it is an important candidate molecule in SLE. IFNβ has distinct features as compared to IFNα: higher affinity binding to the shared IFN-I receptors, IFNβ specific gene transcripts, induction of senescence in fibroblast. As a critical non-hematopoietic component in BM, MSCs create a microenvironment for hematopoiesis and immunity. MSCs display robust immunomodulatory properties. MSC defects have been suggested in autoimmune diseases. Taking into consideration the importance of IFNβ and MSCs in autoimmune diseases, here we set out to investigate the role of IFNβ and MSC in SLE pathogenesis, and the underlying mechanisms. Materials and methods BM MSCs were isolated with FicollPaque gradient centrifugation (1.073 ± 0.001 g/ml) and phenotyped using flow cytometry. Various in vitro approaches including confocal immunofluorescence immunocytochemistry, real-time PCR, western blotting, comet assay, beta-galactosidase assay and RNA interference were applied. Results We compared 6 age paired BM aspirates from healthy controls and SLE patients. SLE MSCs show reduced proliferation rate, increased production of reactive oxygen, and increased DNA damage and repair (DDR), which leads to p53 mediated senescence associate secretory phenotype (SASP) and inhibited immunomodulatory factors production. IFNβ increased 5 folds and IFNβ specific genes are significantly elevated (p < 0.05) in SLE BM MSCs and are closely correlated to the level of Mitochondrial Antiviral Signalling Protein (MAVS) (r > 0.9, p < 0.01), an intracytoplasmic nucleic acid sensor. Silencing MAVs inhibits IFNβ expression and reverses SASP in SLE MSCs. Conclusions SLE is associated with elevated IFN-I in BM. BM MSCs produce IFNβ, have increased DDR and SASP. Thus an IFNβ positive feedback loop forms in SLE BM MSCs. By silencing MAVS, also named Interferon Beta Promoter Stimulator Protein 1, IFNβ expression is inhibited and IFNβ positive feedback loop is disrupted. Moreover, SASP is rescued by MAVs blockage in SLE BM MSCs. Our novel findings of the IFNβ positive feedback loop and related SASP in SLE BM MSCs shed light on SLE pathogenesis. In addition, our study has also revealed the essential role of MAVS in IFNβ positive loop, and thus provided a new potential therapeutic target for SLE treatment.