IL-1 secreting cell assay and its application to cells from patients with rheumatoid arthritis.

The cells within a population that were secreting interleukin-1 (IL-1) were enumerated and visualized by an ELISA-SPOT assay. Initial experiments designed to validate the assay revealed that the number of IL-1 beta spot forming cells was increased by exposing normal blood monocytes to LPS and that spot formation was prevented by incubating the cells with cycloheximide. Normal blood polymorphonuclear leucocytes (PMNs) produced IL-1 alpha and IL-1 beta in response to recombinant granulocyte monocyte colony stimulating factor (rhGMCSF) but not to cytochalasin B, calcium ionophore or LPS. Monocytes and PMN were isolated from the synovial fluid (SF) and blood of patients with rheumatoid arthritis (RA) and the ability of these cells to secrete IL-1 alpha and IL-1 beta compared. A higher proportion of SF derived monocytes were found to secrete IL-1 beta spontaneously compared to the corresponding blood cells. IL-1 alpha secreting monocytes were not detected although high numbers of IL-1 alpha secreting cells were found among cells isolated from rheumatoid synovium. By contrast SF PMNs did not produce IL-1 alpha or IL-1 beta whereas blood PMNs from some (3/8) RA patients produced IL-1 alpha and/or IL-1 beta. It is considered that the IL-1 ELISA-SPOT is a highly sensitive technique for detecting IL-1 secreting cells.

Inhibition of myeloperoxidase by synovial fluid and serum.

An inhibitor of myeloperoxidase has been identified in the synovial fluids and sera from patients with rheumatoid arthritis and sera from normal subjects. Initially, these fluids were found to inhibit stimulus induced degranulation of polymorphonuclear leucocytes independently of the stimulating agent. Subsequently, the fluids were shown to inhibit the released enzyme rather than the degranulation response of polymorphonuclear leucocytes. Both rheumatoid and normal serum samples contained high concentrations of the inhibitor but the concentrations were lower in rheumatoid synovial fluids. The inhibitory activity seemed to be specific for peroxidase as the fluids did not inhibit beta-glucuronidase activity. A protein of relative molecular mass (Mr) 150 kd was purified from synovial fluid by affinity chromatography on myeloperoxidase-Sepharose. It is concluded that serum and synovial fluid contain a novel myeloperoxidase inhibitor, which acts by binding to myeloperoxidase and thereby prevents myeloperoxidase releasing oxidative products in serum.

Identification of a myeloperoxidase inhibitor from normal human serum.

An inhibitor of myeloperoxidase (MPO) has been identified in normal human serum. Initial experiments confirmed that high levels of MPO inhibitory activity are present in sera and that the inhibitor did not act by interfering with the assay. Purification of the inhibitor activity by salt precipitation followed by ion exchange and affinity chromatography revealed the presence of a protein of 150 kD. The purified inhibitory activity displayed dose and time dependency and was not associated with IgG or IgA. It is considered that human serum contains an inhibitor of extracellular MPO capable of protecting against hypohalous acid release in host tissues and that if inhibitor levels are reduced such protection may fail.

A comparison of the effects of urate, hydroxyapatite and diamond crystals on polymorphonuclear cells: relationship of mediator release to the surface area and adsorptive capacity of different particles.

The ability of monosodium urate monohydrate (MSUM), hydroxyapatite and diamond crystals to stimulate phagocytosis, degranulation and secretion of cell movement factors (CMF) from polymorphonuclear leukocytes (PMN) was determined. The ability of each crystal to adsorb PMN derived enzymes and CMF was also compared. MSUM crystals stimulated greater enzyme release and generation of CMF than hydroxyapatite; in contrast, hydroxyapatite crystals exhibited greater adsorption of PMN products. This was partly due to the greater surface area (at a given concentration) of hydroxyapatite crystals. Diamond crystals clearly interacted with PMN, but they did not stimulate degranulation or CMF production.

Oxidative response of polymorphonuclear leucocytes to synovial fluids from patients with rheumatoid arthritis.

Only a minority (7/35, 20%) of synovial fluids from patients with rheumatoid arthritis (RA) and none from patients with other arthritides stimulated the oxidative response of polymorphonuclear leucocytes (PMNs). Superoxide anion generation was measured by superoxide dismutase inhibitable reduction of cytochrome c. The same synovial fluids stimulated superoxide release by PMNs regardless of their source, though they elicited a greater response from RA synovial fluid PMNs than from either RA blood PMNs or blood PMNs from normal subjects. The remaining synovial fluids failed to stimulate any of the PMNs, though some (2/10) stimulated PMNs pretreated with cytochalasin B. The stimulatory activity was removed from RA synovial fluids by protein A-Sepharose and eluted with the void volume on gel chromatography. It is considered that immunoglobulin aggregates in some RA synovial fluids may stimulate the oxidative response of PMNs in the joint space but that most do not because these fluids contain either a specific inhibitor or immunoglobulin aggregates of an inappropriate type, or both.

Concentrations of glycosaminoglycans in synovial fluids and their relation with immunological and inflammatory mediators in rheumatoid arthritis.

The dimethylmethylene blue assay showed higher concentrations of glycosaminoglycans in many synovial fluids from patients with rheumatoid arthritis (RA) than in autologous sera or sera or synovial fluids from normal subjects. These results were taken to suggest that the glycosaminoglycans in RA synovial fluid were abnormally raised and derived from cartilage. To discover what stimulated such glycosaminoglycan release in RA joints relations were sought between synovial fluid concentrations of glycosaminoglycans and immunological and inflammatory mediators. It was shown that RA synovial fluid glycosaminoglycan concentrations correlated with synovial fluid C3d concentrations but not with synovial fluid rheumatoid factor concentrations, polymorphonuclear leucocyte numbers, myeloperoxidase concentrations, or the ability of the synovial fluids to release free radicals from normal polymorphonuclear leucocytes. A correlation was found between synovial fluid C3d and interleukin 1 concentrations as judged by both lymphocyte activating factor activity and immunoassay, but no significant correlation was detected between interleukin 1 and glycosaminoglycan concentrations. It is suggested that in the rheumatoid joint locally produced cytokines, in addition to interleukin 1, together stimulate glycosaminoglycan release from cartilage and render it vulnerable to attack by other processes.

Depressed degranulation response of synovial fluid polymorphonuclear leucocytes from patients with rheumatoid arthritis to IgG aggregates.

No difference was found between the degranulation responses to FMLP of synovial fluid (SF) polymorphonuclear leucocytes (PMNL), from patients with rheumatoid arthritis (RA), and either paired blood PMNL or blood PMNL from a healthy donor. In contrast, the response of SF PMNL to heat-aggregated IgG was often reduced compared with autologous blood PMNL. Similarly, SF from some (35%) RA patients stimulated degranulation of PMNL but the response of SF-derived PMNL to autologous stimulatory SF was reduced compared with the response of blood PMNL. The stimulatory activity of the SF was removed by sepharose-protein A. These results were taken to suggest that the activity is due to immunoglobulin aggregates and that SF PMNL (from some RA patients) are tachyphylactic to stimulation by immunoglobulin aggregates as measured by degranulation because they have been stimulated by immunoglobulin aggregates in vivo. In other studies the concentration of myeloperoxidase (MPO) was measured enzymically in RA SF and was found to be present in varying amounts. However, only a weak relationship was found between MPO levels and either PMNL numbers or levels of complement-bearing IgG aggregates in SF. It is considered that the relationship between MPO and immunoglobulin aggregates levels is obscured by the presence of a peroxidase inhibitor in the fluids and/or because only aggregates bound to tissue stimulate degranulation in vivo.

Recombinant human interleukin-1 beta primes human polymorphonuclear leukocytes for stimulus-induced myeloperoxidase release.

Recombinant human Interleukin-1 (rhIL-1) beta was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhIL-1 beta and then stimulated with either heat-aggregated IgG (Hagg) or N-formyl-methionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 micrograms/ml Hagg, 2.5 X 10(-9) M FMLP) did not stimulate degranulation in the absence of rhIL-1 beta. However, such concentrations elicited marked degranulation from PMNs preincubated with rhIL-1 beta (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1 beta employed and on the period of incubation. In other experiments, the effect of rhIL-1 beta on the PMN oxidative response was determined. rhIL-1 beta did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1 beta may potentiate PMN degranulation, but not their oxidative response.

Enhanced oxidative response of polymorphonuclear leukocytes from synovial fluids of patients with rheumatoid arthritis.

Polymorphonuclear leukocytes (PMN) isolated from the synovial fluid (SF) of patients with rheumatoid arthritis (RA) exhibited an enhanced oxidative response to heat aggregated IgG (Hagg) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as compared with either autologous or normal blood PMN. Normal blood PMN pretreated with synovial fluid showed a significantly increased response to FMLP which was unaffected by prior dialysis of SF. The degree of enhancement produced varied between SF's and was dependent on the period for which cells were incubated and on the concentration of SF used. In contrast there was no enhancement of the oxidative response when Hagg was used as a stimulus. Indeed, SF produced a dose dependent inhibition of Hagg stimulated superoxide production. These observations suggest that SF's from patients with RA contain factors which both enhance and inhibit the oxidative response of PMN depending on the subsequent stimulus.

The effect of biotic and abiotic implants on the recognition of Blatta orientalis cuticular transplants by the cockroach Periplaneta americana.

The immune system of the American cockroach, Periplaneta americana, was activated to recognise as "non self" xenogeneic tissue normally treated as "self". Activation was accomplished by injecting into the insect haemocoele material known to elicit an encapsulation response such as Blaberus craniifer cuticular tissue and 6B-Sepharose beads. In these insects, cuticular skingrafts from Blatta orientalis, a closely related species of cockroach, were rejected by more than half of the recipients. There was no rejection of skingrafts by naive insects showing that the immune system of the cockroach had been triggered non-specifically to recognise tissue previously treated as "self".