C. J. Elson

Alteration of cartilage metabolism by cells from osteoarthritic bone

OBJECTIVE To determine whether bone cells alter cartilage metabolism. METHODS Bone cell cultures were established using explants obtained from the hip and knee joints of 9 patients with osteoarthritis (OA) and 6 subjects without arthritis (nonarthritic [NA]). NA human cartilage biopsy samples were incubated in the presence or absence of bone-derived cells, and the effects on glycosaminoglycan (GAG) release from cartilage were measured. RESULTS Bone cell cultures secreted osteocalcin (OC) and did not contain cells expressing leukocyte common antigen. None of the 8 cultures established from NA bone, compared with 17 of 32 from OA bone, significantly altered GAG release from cartilage (P = 0.006). In knees with medial joint damage, 38% of the cultures derived from the medial side of the joint increased GAG release from cartilage. In contrast, 77% of the cultures derived from the lateral side of the joint had an effect on GAG, with 38% increasing and 38% decreasing GAG release. Seven cytokines were measured in OA bone cell supernatants. No significant difference was apparent in the concentration of any one cytokine when supernatants were compared according to their effects on GAG release. CONCLUSION Bone cells from OA patients can influence cartilage metabolism. This might explain why increased subchondral bone activity can predict cartilage loss.

Immunologically ignorant autoreactive T cells, epitope spreading and repertoire limitation

The factors that may cause antigen-presenting cells to alter the pattern of protein processing and presentation to autoreactive T cells, and thereby stimulate autoimmune disease, are currently under debate. In this article, Chris Elson and colleagues suggest that cytokines associated with T helper 1 (Th1) cells alter the processing of proteins and that this effect can be counteracted by Th2-associated cytokines.

Increased serum C reactive protein may reflect events that precede radiographic progression in osteoarthritis of the knee

OBJECTIVE Raised serum C reactive protein (CRP) and hyaluronate (HA) concentrations predict the progression of knee osteoarthritis (OA) in the long term but the consistency of these relations with time is unknown. The purpose of this work was therefore to determine if raised CRP and HA at entry and three years before entry (−3 years) predict radiological progression of knee OA in a group of patients between entry and five years. METHODS Knee radiographs from 90 patients with knee OA at entry and five years follow up were assessed for progression of disease over five years. The concentrations of serum CRP and HA were measured at entry (n=90) and also in 40 serum samples available from −3 years. Odds ratios (OR) for predicting progression were calculated by logistic regression. RESULTS Serum CRP at entry was not predictive of progression between entry and five years (OR 1.12, 95% CI 0.81, 1.55) but serum CRP at −3 years was predictive of progression (OR 1.90, 95% CI 1.01, 3.28). Serum HA concentration at entry predicted progression between entry and five years (OR 2.32, 95% CI 1.16, 4.66). CONCLUSION These results are consistent with previous reports relating to HA, and suggest that raised serum CRP reflects events that precede a period of later radiographic progression in knee OA. However, because of the large overlap between groups, the serum CRP or HA concentration are not good predictors of individual patient progression and have a poor sensitivity and specificity.

Extended haplotypes and linkage disequilibrium in the IL1R1-IL1A-IL1B-IL1RN gene cluster: association with knee osteoarthritis

The interleukin-1 gene cluster is a key regulator in a number of chronic disease processes. We explored the linkage between nine polymorphic loci in the IL1R1 promoter, eight in the IL1A–IL1B–IL1RN gene complex, and their association with osteoarthritis (OA), a common complex disease associated with low-level inflammation. Using 195 healthy controls, we identified eight novel polymorphisms in the IL1R1 exon 1A region. We found limited LD between IL1R1 and the IL1A–IL1B–IL1RN cluster, although LD within these two individual groups was high. To test association with knee OA, we genotyped 141 patients from Bristol (UK) at the 17 loci. IL1R1 promoter haplotypes showed no association with disease. However, within the IL1A–IL1B–IL1RN complex, we identified a common haplotype conferring a four-fold risk of OA (P=0.00043; Pc=0.0043) and one IL1B–IL1RN haplotype conferring a four-fold reduced risk (P=0.0036; Pc=0.029). To replicate these associations, we subsequently examined 163 knee OA patients from London. Here, the effects of the haplotypes were confirmed: the risk IL1A–IL1B–IL1RN haplotype conferred a two-fold risk of OA (P=0.02), and the protective IL1B–IL1RN haplotype conferred a five-fold reduced risk of OA (P=0.0000008). These results may help to explain the genome-wide scan linkage data and functional observations concerning association between IL-1 and OA.

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60

The hypothesis that T‐cell responses to the 60 000 MW family of heat‐shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon‐γ and those who did not, although patients whose hsp 60‐stimulated T cells produced interleukin‐4 (IL‐4) and/or IL‐10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL‐10 production by hsp 60‐stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60‐stimulated cells produce T‐helper 2 type cytokines.

Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis

We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce auloantibodies to thyroglobulin (Tg) and/or thyroid peroxidasc (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15–25 × I06 cells mouse and the highest levels were recorded 2–3 weeks later. In contrast, Tg and/or TPO antibodies were undctcctablc in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCTD recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donors scrum Tg antibodies. Blood lymphocytes transferred to SCiD recipients were also able to produce Tg antibodies of subclasses1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.

Chondrocyte tumor necrosis factor receptors and focal loss of cartilage in osteoarthritis.

OBJECTIVE Osteoarthritis (OA) is characterized by focal loss of cartilage. Here we show for the first time that tumor necrosis factor (TNF) alpha can act on cartilage but only at specific sites where chondrocyte TNF alpha-receptor (R) expression is high. DESIGN Cartilage explants from specified sites in the knee joints of both OA patients and non-arthritic (NA) subjects were cultured with and without TNF alpha for 14 days and cumulative glycosaminoglycan (GAG) release into the supernatant measured. p55 and p75 TNF-R expression was measured by flow cytometry on chondrocytes isolated from the same sites. RESULTS Cartilage explants from different sites in knee joints from both OA patients and NA subjects varied in their susceptibility to TNF alpha. Overall, the proportion of samples that responded to TNF alpha was higher in cartilage taken from OA joints than cartilage from NA subjects. Variations in p55 and p75 TNF-R expression were found between chondrocytes from different sites. p55, but not p75 TNF-R, expression on chondrocytes was closely related to the susceptibility of explants from the same site to TNF alpha-induced GAG loss. CONCLUSION It is considered that focal loss of cartilage will occur at sites where chondrocyte p55 TNF-R expression is high, if sufficient TNF alpha is present, and that these results identify a mechanism by which cytokine-mediated focal loss of cartilage may occur.

Susceptibility to pristane-induced arthritis is altered with changes in bowel flora

Pristane-induced arthritis (PIA) is unique among the animal arthritides in that a non-infectious, non-antigenic oil induces a chronic immune based arthritis with a prolonged delay between exposure to the inciting agent and development of the disease. Mice with pristane-induced arthritis have elevated T cell and humoral responses to the 65 kDa heat shock protein derived from Mycobacterium bovis (hsp65) and in common with several other models of autoimmune diseases the incidence of PIA is markedly suppressed by preimmunisation with hsp65 in Freunds incomplete adjuvant (Thompson et al. (1990) Eur. J. Immunol. 20, 2479). Recent studies have investigated how autoimmune reactions to heat shock proteins are involved in the development of arthritis. Arthritic CBA/Igb mice given pristane alone develop antibodies to both hsp65 and GroEl (bacterial 60 kDa heat shock proteins) and to hsp58 (the mammalian equivalent). Moreover, the splenic T cells of such mice proliferate vigorously in response to both bacterial and mammalian 60 kDa heat shock proteins. Remarkably, the anti-hsp65 antibody response in normal mice rises rapidly with age, directly correlating with the age related incidence of PIA. In addition, specific pathogen free mice (SPF) maintained in an isolator have negligible anti-hsp65 responses but these convert to positive responses if the animals are exposed to the open part of the animal facility (Thompson et al. (1992) Arthritis Rheum. 35, 139).(ABSTRACT TRUNCATED AT 250 WORDS)

The separation and identification by monoclonal antibodies of dog IgG fractions

Four fractions of IgG from normal dog serum have been successfully isolated by gel filtration followed by protein A and protein G affinity chromatography using the fast protein liquid chromatography (FPLC) system. Protein A chromatography produced three peaks: peak 1 was fallthrough material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contained bound material eluting at pH 3.5. The three peaks were then subjected individually to protein G affinity chromatography. Peak 1 from protein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a single peak at pH 3.8, and was called peak x. Peak 3 from protein A chromatography emerged as two separate peaks (y and z) off the protein G column; peak y bound and eluted at pH 4.1, and peak z bound weakly to protein G and emerged as a broad band at pH 8. Peaks w, x, y and z have been named gamma w, gamma x, gamma y and gamma z, respectively, and there purified IgG fractions were used to immunize mice for the preparation of monoclonal antibodies (McAbs). To date, two sets of McAbs have been produced: one which recognizes an epitope present in both gamma w and gamma z fractions and another set of McAbs which recognizes an epitope in the gamma x and gamma y fractions.

Hydroxyapatite and urate crystal induced cytokine release by macrophages.

Destructive osteoarthritis is characterised by rapidly progressive joint destruction associated with intra-articular deposition of hydroxyapatite crystals. The possible role of such crystals in the pathogenesis of this condition was investigated by testing the ability of hydroxyapatite crystals to stimulate the production of bone resorbing activity from mouse peritoneal macrophages. Urate crystals were used for comparison. Culture supernatants were tested for bone resorbing activity using the mouse calvarial bone resorption assay, for interleukin 1 using a standard lymphocyte activation assay, and for prostaglandin E2 by radioimmunoassay. Culture supernatants from macrophages incubated with hydroxyapatite crystals contained dialysable bone resorbing activity, high concentrations of prostaglandin E2, but no interleukin 1 like activity. The production of the bone resorbing agent was prevented by culturing macrophages with hydroxyapatite crystals in the presence of indomethacin. By contrast, culture supernatants from macrophages incubated with urate crystals contained bone resorbing activity, which was only partly removed by dialysis, and interleukin 1 like activity. The latter was shown to be increased in culture supernatants from macrophages incubated with urate crystals in the presence of indomethacin, while production of bone resorbing activity was partially inhibited. It is considered that the bone resorbing activity liberated from macrophages stimulated by hydroxyapatite crystals can be explained by the presence of prostaglandin E2 alone, whereas the activity liberated by urate crystals is due to both prostaglandin E2 and interleukin 1.