Warren D. Blackburn

Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain‐ specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three‐armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophos‐ phate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.

Cartilage imaging in osteoarthritis

Osteoarthritis (OA) is the most common articular disorder encountered worldwide. Its successful evaluation (and eventual treatment) depends on establishing a set of criteria for measuring disease progression. An ideal measurement would evaluate changes in articular cartilage, where the primary pathology of the disease takes place. Plain radiographs are the simplest and most readily employable means of joint evaluation, and now microfocal radiographs have been developed, which magnify the radiograph and help portray the joint space more accurately. However, radiography, along with nuclear medicine scans, arthrography, and computed tomography (CT) scans, are limited in their use because they are unable to detect early cartilage abnormalities. Magnetic resonance imaging (MRI) has the advantages of multiplanar imaging, soft tissue contrast, and noninvasiveness. Like radiography, MRI can underestimate the extent of cartilage abnormality. The most sensitive technique for measuring superficial articular abnormalities is arthroscopy, and small-bore arthroscopes are being used to assess knee damage in conscious, nonsedated patients. However, it is not yet clear if arthroscopy can detect subtle changes over time, and vision can be blocked by cloudy synovial fluid. Finally, although it is usually well tolerated, arthroscopy is an invasive technique.

Eosinophilia myalgia syndrome.

The term eosinophilia myalgia syndrome (EMS) was coined in 1989 after a cluster of cases with symptoms of incapacitating myalgias and eosinophilia were reported. This syndrome has been only defined as varying degrees of myalgias and peripheral eosinophilia. Case reports of EMS are protean and do not show a consistent clinical picture, raising the question of whether this reflects a single disorder or is a conflation of various disorders. There have been only two studies evaluating the association of EMS with 1-tryptophan. These two included only 23 patients with EMS. Apart from the obvious statistical fragility inherent in such small studies, each is further weakened by differences in the mechanisms by which patients and controls were selected. Furthermore, the continued reports of EMS after 1-tryptophan was removed from the market raise additional questions about the association. Nonetheless, there has been an inordinate reliance on a history of 1-tryptophan ingestion in making the diagnosis of EMS. When presented case studies, clinicians were much more likely to make the diagnosis of EMS when a history of 1-tryptophan ingestion was included. These observations underscore the need for careful application of well-considered diagnostic criteria to the study of new syndromes and their potential associations.

Management of osteoarthritis and rheumatoid arthritis: Prospects and possibilities

Conventional drug therapy in rheumatoid arthritis (RA) has failed to control the longterm morbidity and mortality associated with RA. Similarly, drug therapy for osteoarthritis (OA) can relieve symptoms, but it is not clear that it alters progression of disease. Three classes of drugs are widely used for treatment of RA: nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and the slow-acting agents. In most patients, pharmacologic therapy is initiated with NSAIDs. These drugs can relieve symptoms but do not alter the course of the disease. The gastrointestinal and other side effects attributed to these compounds are well known. Similarly, use of corticosteroids can provide rapid pain relief to patients with RA and, if used in low doses, pose limited risk of toxicity. Slow-acting agents, including gold, d-penicillamine, and methotrexate, appear to decrease radiographic progression and improve clinical and biochemical indicators of RA. Therefore, newer treatment philosophies encourage use of slow-acting agents earlier in the course of the disease in order to prevent or diminish bone and joint erosions and destruction and other manifestations of disease progression. Drugs under investigation for the treatment of arthritis appear to exhibit disease-modifying or immunomodulating properties. Tenidap is a novel agent that possesses a dual mechanism of action: cyclooxygenase inhibition and modulation of cytokine activity. In addition, several biologic agents, including antibodies to tumor necrosis factor-alpha (TNF-alpha) and to intercellular adhesion molecule-1, may prove useful. These immunotherapeutic strategies are based on knowledge of the role of cytokines in the inflammatory process in arthritis. Osteoarthritis may be managed using drug and nondrug modalities. Weight loss is especially important when OA is in the weight-bearing joints. Biopsies of synovium from patients with OA show evidence of inflammation, but whether this disease should be treated with analgesics alone or with anti-inflammatory drugs remains controversial. Other treatment modalities, including tissue transplants and cytokine-modulating drugs, are emerging for the potential therapy of OA. Surgery may also be appropriate if drug treatment fails to control symptoms.

Determinants of neutrophil HOCl generation: ligand-dependent responses and the role of surface adhesion.

Neutrophil (PMN) generation of HOCI, an oxidant important in mediating tissue injury by PMN proteases, requires PMN production of H2O2 and the catalytic activity of myeloperoxidase (MPO). Production of H2O2 and MPO release vary with the PMN activating ligand and are facilitated by cellular adhesion. Leukotriene B4, platelet‐activating factor, heat‐aggregated immunoglobulin G (HAIgG), tumor necrosis factor α (TNF‐α), and f‐Met‐Leu‐Phe (fMLP) all triggered significant superoxide production but negligible H2O2 or HOCI generation when added to suspended PMNs. Production of H2O2 was observed when fMLP, TNF‐α, or HAIgG was added to PMNs adherent to bovine serum albumin (BSA)‐coated wells, but significant production of HOCI was observed only when HAIgG was added to PMNs adherent to BSA‐coated wells or when suspended PMNs treated with TNF‐α were allowed to settle in BSA‐coated wells. Even greater production of both H2O2 and HOCI was observed when PMNs were incubated in wells coated with IgG (SAIgG). HOCI generation, when observed, was accompanied by release of MPO. Nonadherent PMNs generated HOCI when treated with 50–100 ng/ml phorbol myristate acetate or when stimulated with fMLP following treatment with cytochalasin B; PMN activation under these conditions was also associated with MPO release but HOCI production was much less efficient relative to PMNs stimulated by SAIgG. These studies indicate that surface adhesion and ligand‐induced responses that facilitate release of myeloperoxidase and dismutation of superoxide to H2O2 are required for production of extracellularly released HOCI; these responses are most efficiently utilized during PMN interaction with SAIgG. J. Leukoc. Biol. 56: 654–660; 1994.

Evaluation of patients with back pain of suspected inflammatory nature.

PURPOSE Detection of early inflammatory back disease is often difficult. Certain clinical characteristics have been reported to increase the likelihood of its detection in referral patients, but the usefulness of these clinical characteristics has not been evaluated in an open population. In our study, we undertook to evaluate the value of the clinical history as a screening test for inflammatory back disease in a general population. PATIENTS AND METHODS Twenty-three male patients with back pain of moderate duration and with clinical characteristics suggestive of inflammatory back disease were recruited by advertising and were studied by various means, including computed tomography (CT), scintigraphy, and radiography. RESULTS One patient had radiographic sacroiliitis. Two had positive results for the B27 antigen, and another had positive results for the cross-reacting HLA antigen B7. Eight patients had abnormal scintiscans of the sacroiliac joints. Twenty-one of 23 patients and 20 of 23 control subjects had abnormalities that were detected by CT. Repeat plain radiographs of the pelvis done 36 months after enrollment into the study did not uncover further evidence of sacroiliitis. CONCLUSIONS These results indicate plain radiographic evidence of sacroiliitis will often not develop in patients with historical features suggestive of inflammatory back disease even with long-term evaluation, thus vitiating the specificity of these historical findings in men with back pain of relatively brief duration.

Lack of evidence of systemic inflammatory rheumatic disorders in symptomatic women with breast implants

&NA; Breast implants containing silicone have been used for approximately 30 years for breast augmentation or reconstruction. In general, the implants have been well tolerated and reports have indicated a high degree of patient satisfaction. Nonetheless, there have been anecdotal reports of patients with musculoskeletal complaints that have been attributed to silicone breast implants. To investigate this further, we prospectively examined 70 women with silicone breast implants who had complaints that they or their referring physicians thought were related to their implants. On clinical examination, the majority of the patients had fibromyalgia, osteoarthritis, or soft‐tissue rheumatism. One patient had rheumatoid arthritis, which predated her implants, and one had Sjögrens syndrome. Because many of our patients had myalgic symptoms, we further evaluated these patients by measuring circulating levels of soluble factors including interleukin‐6, interleukin‐8, tumor necrosis factor‐alpha, soluble intercellular adhesion molecule‐1, and soluble interleukin‐2 receptor, which have been previously found to be elevated in patients with inflammatory diseases. We found that the levels of these molecules in women with silicone breast implants were not different from those seen in normal subjects and were significantly less than those seen when examining chronic inflammatory disorders such as rheumatoid arthritis or systemic lupus erythematosus. In summary, our clinical and laboratory evaluation of symptomatic breast implant patients argues against an association of silicone breast implants with a distinctive rheumatic disease or a systemic inflammatory disorder. Given these findings and the clinical picture, it is our impression that most symptomatic women with silicone breast implants have well‐delineated noninflammatory musculoskeletal syndromes. Moreover, these data fail to support the concept that their symptoms are due to a systemic inflammatory response related to their implants.

Solid-phase radioimmunoassay for human neutrophil elastase: A sensitive method for determining secreted and cell-associated enzyme☆

A solid-phase radioimmunoassay for measuring neutrophil elastase in the range 0.08-4 ng/ml has been developed. A monospecific, precipitating antibody capable of inhibiting elastinolysis was produced by repeated immunizations of a goat. The IgG fraction and affinity-purified antibodies of this serum were then obtained and used to develop this radioimmunoassay. There was no cross-reactivity in binding of the radiolabeled antisera with lactoferrin, cathepsin G, or serine proteinases with amino-terminal amino acid sequence homology. Although serum influences the measurement of catalytically active neutrophil elastase when compared to diisopropylfluorophosphate-treated neutrophil elastase, antigenic elastase may still be measured in body fluids. Furthermore, this assay is more sensitive than commercially available substrates used for quantitating neutrophil elastase by functional activity. We have found this quantitative assay extremely useful in balance studies to measure secreted and cell-associated elastase and in screening of biological fluids for the presence of the enzyme.

Characterization of a SV40-transformed rheumatoid synovial fibroblast cell line which retains genotypic expression patterns: A model for evaluation of anti-arthritic agents

SummaryA chimeric Adenovirus-Simian Virus 40 (AdSV40) containing the large T antigen was used to transform rheumatoid synovial fibroblasts. A rheumatoid synovial fibroblast cell line was established by infection of primary rheumatoid arthritis (RA) synovial fibroblasts at Passage 10 with AdSV40 recombinants followed by selection in semisoft agarose cultures. The transformed cells grew anchor independent, exhibited continuous proliferation (>65 passages) in monolayer culture, and formed multiple visible foci.The transformed synovial fibroblasts showed expression of the simian virus 40 large T antigen in the nucleus as determined by immunofluorescence staining. In addition, indirect immunofluorescence staining demonstrated that the transformed cells stained specifically with a fibroblast-specific antibody 1B10. Studies involving expression of metalloproteinases showed that collagenase and stromelysin were induced by phorbol 12-myristate 13-acetate (PMA), and such an induction was repressed by dexamethasone typical of primary RA fibroblasts. Levels of mRNAs for IL-1β, TNF-α, and c-jun were increased by PMA, and the mRNA transcripts of these genes were also repressed by addition of dexamethasone to the culture media. Our results indicate that transformed RA synovial fibroblasts display a similar gene expression pattern in response to PMA and dexamethasone as observed for untransformed primary RA synovial fibroblasts. These transformed rheumatoid arthritis synovial fibroblast cells provide an ideal cell culture model in which to test the efficacy of novel arthritis gene therapy reagents.

HOCl production by human neutrophils activated by surface-associated IgG: requirement for influx of extracellular calcium.

Neutrophils produce large quantities of HOCI when stimulated by surface‐associated immunoglobulin G, a result not seen when neutrophils are stimulated with soluble complexes of IgG. Compared with unactivated cells or cells stimulated with soluble aggregates of IgG, a significant influx of extracellular 45Ca2+ was observed in cells activated by surface‐associated IgG. Removal of extracellular calcium with EGTA almost completely blocked HOCI production. Similarly, treatment of neutrophils with lanthanum, which has been shown to interfere with calcium channels, also effectively blocked HOCI production. These results were not secondary to an overall decrease in activation, as superoxide production and release of the specific granule protein lactoferrin and the azurophilic granule protein myeloperoxidase were not significantly altered by lanthanum or EGTA. Production of H2O2, the precursor of HOCI, was similarly decreased by both EGTA and lanthanum. Induction of extracellular calcium influx with a calcium ionophore in the presence of soluble aggregates of IgG resulted in HOCI production. Production of HOCI is not sensitive to inhibition by pretreatment of cells with pertussis toxin. These observations indicate that the differences in the biological responses of human neutrophils to surface‐associated IgG compared with soluble aggregates of IgG are associated with differing signaling events, including influx of extracellular calcium. J. Leukoc. Biol. 55: 793–797; 1994.