lactoferrin and Arthritis

We have recently shown that resistin is a key mediator of arthritis accumulating in the inflamed joints and exerting its pro-inflammatory properties independently of TNFalpha. Here we evaluate neutrophils as a cellular source of resistin. Human neutrophils were subjected to subcellular fractionation where the presence of resistin was assessed using western blot, ELISA, and mass spectrometry. Presence of resistin on the neutrophil surface was visualized by flow cytometry. More than 95% of the neutrophils in circulation and in synovial fluid express resistin on their surface. Stimulation of mature neutrophils with fMLF induced release of resistin into supernatants and increased expression of resistin on the surface. Resistin is mobilized simultaneously with lactoferrin, a protein found in specific granules, and with granule-stored CR3/CD11b. Subcellular fractionation of human neutrophils demonstrated the presence of resistin in azurophilic and in specific granules. Here we show that neutrophils have two pools of resistin, the major one exists in specific granules, and the second on their cell membrane. Release of resistin from the neutrophil granules probably serves the main source of resistin at the site of inflammation.

Topics: Adult; Aged; Arthritis; CD11b Antigen; Cell Membrane; Female; Gene Expression Regulation; Humans; Inflammation; Joints; Lactoferrin; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Resistin; Secretory Vesicles

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [(3)H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-DL: -penicillamine (SNAP) (500 microM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [(3)H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.

Topics: Animals; Annexin A5; Apoptosis; Arthritis; Caspase 3; Caspases; Cell Proliferation; Cells, Cultured; Collagen Type II; Enzyme Activation; Fibroblasts; Lactoferrin; Male; Nitric Oxide; Oligopeptides; Peptide Fragments; Poly(ADP-ribose) Polymerases; Protein Binding; Rats; Rats, Wistar; Synovial Fluid

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteolytic enzymes, which degrade several components of extracellular matrix, in arthritic synovial cells. In cultured synovial fibroblasts, both nitric oxide (NO) and reactive oxygen species (ROS) are potent inducers of MMPs production. PEP1261, a tetrapeptide derivative used in this study, corresponds to residues of 39-42 human lactoferrin. The parent protein lactoferrin is able to inhibit the production of free radicals in rheumatoid joints and it regulates many aspects of inflammation. This study is aimed to examine the effects of PEP1261 on MMP-2 production in the presence of nitric oxide donor in cultured synovial fibroblasts from collagen-induced arthritic rats. PEP1261 affects a significant reduction in nitrite levels as well as in MMP-2 production in SNAP stimulated synovial fibroblasts and this is validated by gelatin zymography and immunoblot analysis. Furthermore, RTPCR analysis has demonstrated that PEP1261 inhibits MMP-2 mRNA expression in SNAP treated synovial fibroblasts. The results of this study suggest that PEP1261 possesses antiarthritic activity by inhibiting nitrite levels as well as MMP-2 expression better than control peptides viz., KRDS and RGDS.

Topics: Animals; Arthritis; Collagen; Disease Models, Animal; Fibroblasts; Inhibitory Concentration 50; Lactoferrin; Matrix Metalloproteinase 2; Nitric Oxide; Peptide Fragments; Peptides; Rats; Rats, Wistar; RNA; Synovial Membrane

To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis.. Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days.. DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival.. Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

Topics: Administration, Topical; Animals; Arthritis; Arthritis, Infectious; Collagen; Disease Models, Animal; Injections, Intra-Articular; Joints; Lactoferrin; Male; Mice; Mice, Inbred DBA; Staphylococcal Infections; Staphylococcus aureus; Tissue Distribution

We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in SF was due to the concerted action of both reactive oxygen species and lysosomal proteinases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; alpha 1-Antitrypsin; alpha-Macroglobulins; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Female; Humans; Joints; Lactoferrin; Male; Middle Aged; Neutrophils; Osteoarthritis; Pancreatic Elastase; Synovial Fluid

The mass fraction of certain elements was measured in isolated granulocytes and isolated granulocyte granule fractions from patients with active inflammatory arthritides (N = 6) and healthy controls (N = 6). The patients had significantly increased amounts of Ca in the granulocytes, in the specific and light azurophil granules, but normal Ca amounts in the dense azurophil granules. Sr was below the detection limit in the granulocytes and granule fraction from controls, but it appeared in high concentrations in the granulocytes and all granule fractions from the patients. The patients had considerably increased granulocyte amounts of Mn but only slightly increased Mn concentrations in the specific granules. Mn was not detectable in azurophil granules from patients and controls. A prominent accumulation of Fe was seen in the granulocytes from the patients, together with an Fe accumulation in the specific granules. Fe was below the detection limit in azurophil granules from patients and controls. The patients had reduced granulocyte Zn and reduced amounts of Zn in the dense and light azurophil granules but normal Zn amounts in the specific granules. The results obtained indicate that the granulocyte accumulation of Ca, Sr, and Fe observed during chronic inflammation is associated with corresponding granule accumulation of these metals; the considerable Mn accumulation in granulocytes during inflammation is not localized in their granules; and the granule subpopulations differ in their capacity to store certain metals.

Topics: Arthritis; Chronic Disease; Cytoplasmic Granules; Humans; Lactoferrin; Metals; Neutrophils; Peroxidase; Spectrometry, X-Ray Emission

Some 40% of knee-joint synovial fluids from arthritic patients show the presence of bleomycin-detectable iron. This is released from a protein component of the fluid to bleomycin at acidic pH values. Patients whose fluids release iron have lower contents of transferrin, lactoferrin and caeruloplasmin than do patients whose fluids do not release iron to bleomycin. These proteins are important extracellular antioxidants, and measured antioxidant activities are extremely low in the iron-releasing fluids. The propensity of some fluids to release iron at low pH values, characteristic of the microenvironment beneath adherent macrophages, coupled with their decreased antioxidant protection against iron-stimulated oxygen-radical damage, might explain previously reported correlations between clinical disease severity, lipid peroxide content and the presence of bleomycin-detectable iron [Rowley, Gutteridge, Blake, Farr & Halliwell (1984) Clin. Sci. 66, 691-695].

Topics: 2,2'-Dipyridyl; Arthritis; Bleomycin; Ceruloplasmin; Copper; Humans; Hydrogen Peroxide; In Vitro Techniques; Iron; Lactoferrin; Lactoglobulins; Proteins; Synovial Fluid; Transferrin

Eosinophils are seldom noted in inflammatory synovial fluids but are reported to infiltrate the synovial tissue in inflammatory arthritides. To elucidate a possible role for eosinophils in inflammatory joint reactions the concentrations of eosinophil cationic protein (ECP)--a specific granule protein from eosinophils--were measured by radioimmunoassay in 90 synovial fluids from patients with various inflammatory arthritides (rheumatoid arthritis, reactive and crystal arthritides, Reiter's disease and psoriatic arthropathy). In the same specimens lactoferrin was measured as an indicator of neutrophil-involved inflammation. In comparison with the normal circulating levels of ECP and lactoferrin the measured synovial fluid concentrations of both proteins were considerably raised in all patient groups with inflammatory joint diseases in contrast to patients with non-inflammatory arthritides. There was a striking positive correlation between the ECP and lactoferrin synovial fluid concentrations. These data indicate that eosinophil activation is prominent in inflammatory joint reactions and is linked to the activation of neutrophils. The regulation of degranulation or secretion by eosinophils is unknown. Our in-vitro studies showed that peripheral blood isolated neutrophils as well as eosinophils degranulated when exposed to IgG complexes. However, eosinophil degranulation was modest compared with neutrophil degranulation. These data suggest that neutrophil phagocytosis of, for example, immune complexes may be one major mechanism in neutrophil degranulation but that other factors determine the appearance of eosinophil products in inflammatory synovial effusions. The possible modulatory or harmful role of eosinophils in inflammatory joint disease can at present only be speculated on.

Topics: Adolescent; Adult; Aged; Arthritis; beta-Thromboglobulin; Blood; Blood Proteins; Eosinophil Granule Proteins; Female; Granulocytes; Humans; Immunoglobulin G; Lactoferrin; Male; Middle Aged; Ribonucleases; Synovial Fluid

Topics: Arthritis; Humans; Iron; Lactoferrin; Lactoglobulins; Pancreatitis