H. Patrick McNeil

A Phospholipid-β2-Glycoprotein I Complex Is an Antigen for Anticardiolipin Antibodies Occurring in Autoimmune Disease But Not with Infection

Anticardiolipin antibodies (aCL) purified from patients with autoimmune disease have recently been shown to interact with a phospholipid-binding plasma protein, β2-glycoprotein I (β2-GPI). The aim of this study was to determine whether aCL purified from patients with infection also interact with β2 -GPI. aCL purified from 23 patients with malaria, infectious mononucleosis, tuberculosis, hepatitis A or syphilis did not require the presence of β2-GPI to bind cardiolipin (CL). In contrast, aCL were purified from 11 out of 12 patients with autoimmune disease that bound CL only in the presence of β2-GPI. Thrombotic complications appear to be associated with aCL occurring in autoimmune disease but not with aCL associated with infections. We postulate that this increased risk of thrombosis in the autoimmune group may be due to the presence of aCL that bind CL in association with β2-GPI, a plasma protein with anticoagulant activity.

Mast cell activation and migration to lymph nodes during induction of an immune response in mice.

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.

Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics

Autoantibodies to phospholipid antigens can be characterized using solid phase immunoassays to detect anticardiolipin antibodies (ACA) or in phospholipid‐dependent clotting tests where lupus anticoagulant (LA) activity can be demonstrated. It has not been established whether each activity is due to the same or separate antibody subgroups. Plasma from two patients with high levels of both activities were used for purification of ACA and LA using sequential ion‐exchange, gel‐filtration, and anti‐Ig affinity chromatography. Plasma could be separated into fractions containing each activity in the absence of the other. In these fractions, antibodies responsible for LA activity do not bind to isolated phospholipids in solid phase immunoassays, and conversely antibodies binding in these assays (ACA) do not possess LA activity, suggesting LA are directed against a more complex lipid epitope. In addition, in one patient ACA was of IgG isotype only, whilst LA was due to IgG and IgM isotypes. In this patient, the IgG‐ACA was heterogenous with three peaks clearly separated on ion‐exchange chromatography. Affinity purified antiphospholipid antibodies have been previously prepared from a number of patients using a phospha‐tidyl‐serine column and antibodies purified in this manner possess ACA but not LA activity. Taken together, these data indicate that tests for ACA and LA define separable subgroups of phospholipid binding antibodies, thus explaining the discordance often seen between the two activities.

Mast cells contribute to autoimmune inflammatory arthritis via their tryptase/heparin complexes.

Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase β (hTryptase-β) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-β/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.

S100 Calgranulins in inflammatory arthritis

Calgranulins comprise three proteins, S100A8 (Calgranulin A), S100A9 (Calgranulin B) and S100A12 (Calgranulin C) that are predominantly expressed by neutrophils, monocytes and activated macrophages. These S100 calcium‐binding proteins are important molecular mediators in a range of diseases, including inflammatory arthritis, atherosclerosis and microbial infections. Much of the literature has focused on the pro‐inflammatory functions of calgranulins, and this has tended to underplay important regulatory, anti‐oxidant and protective properties. S100A8 and S100A9 are particularly complex in their actions because they exert intracellular and extracellular functions, they form a heterocomplex, S100A8–A9 (calprotectin), and have actions that are independent of or dependent on heterocomplex formation. In some circumstances S100A9 appears to regulate, rather than synergize with the actions of S100A8 and vice versa. Moreover, these calgranulins also bind zinc and other divalent cations and are sensitive to post‐translational oxidative modifications, properties that also affect some functions. It is important to note that S100A8 has potent anti‐oxidant activity, which could be important in host protection. Furthermore, although the genes for S100A8 and S100A9 are induced by activation of the toll‐like receptor/interleukin‐1 pathway, their expression is enhanced by interleukin‐10 and glucocorticoids, thus suggesting a regulatory role in inflammation. On the other hand, S100A12 appears to be predominantly pro‐inflammatory, particularly by its ability to activate mast cells. Measurement of S100A12 levels may be a highly sensitive biomarker for inflammatory disease activity. This review summarizes the current understanding of the biology of calgranulins, with a focus on their pleiotropic roles in inflammatory arthritis.

Human Mast Cell-Derived Gelatinase B (Matrix Metalloproteinase-9) Is Regulated by Inflammatory Cytokines: Role in Cell Migration

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-α significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-γ inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-α significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-γ, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.

Serum Amyloid A Induces Monocyte Tissue Factor

C-reactive protein (CRP) and serum amyloid A (SAA) increase in the blood of patients with inflammatory conditions and CRP-induced monocyte tissue factor (TF) may contribute to inflammation-associated thrombosis. This study demonstrates that SAA is a potent and rapid inducer of human monocyte TF. SAA induced TF mRNA in PBMC within 30 min and optimal procoagulant activity within 4 h, whereas CRP (25 μg/ml)-induced activity was minimal at this time. Unlike CRP, SAA did not synergize with LPS. Procoagulant activity was inhibited by anti-TF and was dependent on factors VII and X, and TF Ag levels were elevated on CD14+ monocytes. Responses were optimal with lymphocytes, although these were not obligatory. Inhibitor studies indicate activation of NF-κB through the ERK1/2 and p38 MAPK pathways; the cyclo-oxygenase pathway was not involved. SAA-induced TF was partially inhibited by high-density lipoprotein, but not by low-density lipoprotein or by apolipoprotein A-I. SAA is a ligand for the receptor for advanced glycation end products (RAGE), and TF generation was suppressed by ∼50% by a RAGE competitor, soluble RAGE, and by ∼85% by anti-RAGE IgG. However, another RAGE ligand, high mobility group box-1 protein, capable of inducing monocyte chemotactic protein-1 mRNA in 2 h, did not induce TF within 24 h. Cross-linking studies confirmed SAA binding to soluble RAGE. Elevated SAA is a marker of disease activity in patients with rheumatoid arthritis, and PBMC from patients with rheumatoid arthritis were more sensitive to SAA than normals, suggesting a new link between inflammation and thrombosis.

Mast cell-restricted tryptases: Structure and function in inflammation and pathogen defense

Mast cells (MCs) are highly specialized immune cells present in mammals and in lower organisms that predate the development of adaptive immunity. The strong evolutionary pressure to retain MCs for >500 million years suggests critical roles for these cells in our survival. In support of this conclusion, no human has been identified to date that lacks MCs, despite the adverse roles of MCs in systemic anaphylaxis and varied inflammatory disorders. MCs express numerous lineage-restricted neutral proteases, and four members of the chromosome 17A3.3 family of tryptases are preferentially expressed in mouse MCs. The anatomical location of MCs at host-environment interfaces has raised the possibility that some of these enzymes are evolutionally conserved because they are needed for combating infectious organisms. Here we review recent insights into the structure and function of MC tryptases in inflammation and host defense against bacteria and other infectious organisms.

An innovative outcomes-based medical education program built on adult learning principles

An innovative medical curriculum at the University of New South Wales (UNSW) has been developed through a highly collaborative process aimed at building faculty ownership and ongoing sustainability. The result is a novel capability-based program that features early clinical experience and small-group teaching, which offers students considerable flexibility and achieves a high degree of alignment between graduate outcomes, learning activities and assessments. Graduate capabilities that focus student learning on generic outcomes are described (critical evaluation, reflection, communication and teamwork) along with traditional outcomes in biomedical science, social aspects, clinical performance and ethics. Each two-year phase promotes a distinctive learning process to support and develop autonomous learning across six years. The approaches emphasize important adult education themes: student autonomy; learning from experience; collaborative learning; and adult teacher–learner relationships. Teaching in each phase draws on stages of the human life cycle to provide an explicit organization for the vertical integration of knowledge and skills. A learning environment that values the social nature of learning is fostered through the programs design and assessment system, which supports interdisciplinary integration and rewards students who exhibit self-direction. Assessment incorporates criterion referencing, interdisciplinary examinations, a balance between continuous and barrier assessments, peer feedback and performance assessments of clinical competence. A portfolio examination in each phase, in which students submit evidence of reflection and achievement for each capability, ensures overall alignment.

The mouse mast cell-restricted tetramer-forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis.

OBJECTIVE Increased numbers of mast cells (MCs) that express beta tryptases bound to heparin have been detected in the synovium of patients with rheumatoid arthritis (RA). The corresponding tryptases in mice are mouse MC protease 6 (mMCP-6) and mMCP-7. Although MCs have been implicated in RA and some animal models of arthritis, no direct evidence for a MC-restricted tryptase in the pathogenesis of inflammatory arthritis has been shown. We created transgenic mice that lack heparin and different combinations of mMCP-6 and mMCP-7, to evaluate the roles of MC-restricted tryptase-heparin complexes in an experimental model of arthritis. METHODS The methylated bovine serum albumin/interleukin-1beta (mBSA/IL-1beta) experimental protocol was used to induce inflammatory monarthritis in different mouse strains. Mice were killed at the time of peak disease on day 7, and histochemical methods were used to assess joint pathology. RESULTS Arthritis was induced in the knee joints of mBSA/IL-1beta-treated mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(+) C57BL/6 mice, and numerous activated MCs that had exocytosed the contents of their secretory granules were observed in the diseased mice. In contrast, arthritis was markedly reduced in heparin-deficient mice and in mMCP-6(-)/mMCP-7(-) C57BL/6 mice. CONCLUSION MC-derived tryptase-heparin complexes play important roles in mBSA/IL-1beta-induced arthritis. Because mMCP-6 and mMCP-7 can compensate for each other in this disease model, the elimination of both tryptases is necessary to reveal the prominent roles of these serine proteases in joint inflammation and destruction. Our data suggest that the inhibition of MC-restricted tryptases could have therapeutic potential in the treatment of RA.