lactoferrin and Vasculitis

Antineutrophil cytoplasmic antibodies (ANCAs) against myeloperoxidase (MPO), proteinase 3 (PR-3), lactoferrin (LF), cathepsin G (CG) and elastase (EL) were determined to investigate whether the presence of ANCAs is closely related to extra-articular manifestations in Japanese patients with rheumatoid arthritis (RA). Antibodies against MPO, PR-3, LF, CG and EL were determined in sera from 125 patients with RA and 83 sera from patients with other rheumatic diseases by enzyme-linked immunosorbent assay. Clinical manifestations and laboratory parameters of the patients were studied from medical records. Thirty of the 125 (24.0%) RA patients were positive for ANCAs for at least one of these 5 ANCA antigens. Among the 5 ANCAs, anti-LF antibody (anti-LF) (16.8%) was most commonly observed in patients with RA. A higher joint score (JS) and an elevated ESR were demonstrated in ANCA-positive RA patients compared to those of ANCA-negative patients (40.8 ± 43.3, 24.3 ± 26.2, p < 0.05, 44.4 ± 22.4, 28.9 ± 23.6, p < 0.05, respectively). No statistical differences in the presence of interstitial pneumonia, cutaneous vasculitis, rheumatoid nodules and mononeuropathy multiplex were observed between ANCA-positive and ANCA-negative patients. The presence of anti-LF is expected to be of pathological relevance, as the action of anti-LF towards LF results in the inhibition of the anti-inflammatory activity of LF.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Antineutrophil Cytoplasmic; Arthritis, Rheumatoid; Arthrography; Biomarkers; Cathepsin G; Enzymes; Female; Humans; Joints; Lactoferrin; Male; Middle Aged; Myeloblastin; Pancreatic Elastase; Peroxidase; Vasculitis; Young Adult

Lactoferrin has repeatedly been proposed to be a target for antineutrophil perinuclear cytoplasmic antibodies (P-ANCA), which are present in 67% of ulcerative colitis (UC) cases. However, this high prevalence has not been achieved with either Western blots or monospecific ELISA on the basis of purified lactoferrin bound to the solid phase. We reevaluated autoantibodies against lactoferrin by indirect immunofluorescence (IIF), using a lactoferrin-tuned granulocyte substrate. Slides with ethanol-fixed human granulocytes were stripped of their P-ANCA targets by high-salt treatment and then reconstituted with human lactoferrin (LFR granulocytes). The slides were then subjected to IIF with a panel of sera (39 UC, 10 antimyeloperoxidase-positive vasculitis, 50 healthy blood donors). The human sera were also analyzed with antilactoferrin ELISA. In 28 of 39 (71.8%) sera from UC patients, antibodies could be determined that bound exclusively to LFR granulocytes. Nuclease-treated cells failed to show this reactivity. ELISA detected antilactoferrin antibodies in only two UC sera. Lactoferrin is a major P-ANCA target in UC but requires DNA to present the epitopes relevant for the reaction with the autoantibodies. Antigen-stripped and lactoferrin-reconstituted granulocytes can be used in IIF to diagnose antilactoferrin antibodies in UC more reliably than with existing ELISA systems.

Topics: Antibodies, Antineutrophil Cytoplasmic; Colitis, Ulcerative; DNA; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Granulocytes; Humans; Lactoferrin; Protein Binding; Vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.

Topics: Adolescent; Adult; Aged; Anemia, Hemolytic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Antibody Specificity; Antimicrobial Cationic Peptides; Autoantibodies; Blood Proteins; Cathepsin G; Cathepsins; Child; China; Colitis, Ulcerative; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Granulomatosis with Polyangiitis; Humans; Lactoferrin; Leukocyte Elastase; Male; Membrane Proteins; Middle Aged; Myeloblastin; Peroxidase; Seasons; Serine Endopeptidases; Sex Factors; Vasculitis

ANCA are autoantibodies directed against polymorphonuclear cell antigens, mainly proteinase 3 (PR3) and myeloperoxidase (MPO), which are implicated in the pathogenesis of small-vessel necrotizing vasculitis. Alpha1-antitrypsin is the main inhibitor of neutral serine proteinase [i.e. human leukocyte elastase (HLE) and PR3] present in PMN alpha-granules (alphaGr). An association first reported by us between PR3 ANCA and the deficient PiZZ phenotype in ANCA-positive systemic vasculitis, now widely confirmed by others, led us to study the incidence and specificity of ANCA among PiZZ subjects.. We tested a population of 191 PiZZ (273 sera) for ANCA activity versus 272 PiMM matched control subjects using alphaGr or antigen-specific ELISA [PR3, HLE, MPO, lactoferin (LF) and bactericidal/ permeability increasing protein (BPI)].. The incidence of antibodies directed against alphaGr and HLE but not PR3, MPO, LF or BPI was increased in the PiZZ as compared to the PiMM group (Fisher probability respectively P < 0.0001 and P < 0.05).. ANCA not directed against classical antigens (MPO and PR3) may be found in PiZZ patients. However, these patients do not develop systemic vasculitis features. Therefore, alpha1-antitrypsin deficiency is not sufficient to induce ANCA positive vasculitides, and may only act as a second hit amplifying factor.

Topics: Adult; Aged; alpha 1-Antitrypsin Deficiency; Antibodies, Antineutrophil Cytoplasmic; Antibody Specificity; Antimicrobial Cationic Peptides; Blood Proteins; Case-Control Studies; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Homozygote; Humans; Infant; Lactoferrin; Leukocyte Elastase; Male; Membrane Proteins; Middle Aged; Myeloblastin; Peroxidase; Phenotype; Serine Endopeptidases; Vasculitis

We isolated a 27-kD protein using cation exchange chromatography from an acid extract of neutrophil granules. N-terminal amino acid sequence analysis of the first 10 residues showed that this protein is azurocidin, a member of the family of neutral serine proteinase found in the neutrophil, which shares amino acid sequence homology with the three other neutral serine proteinases, elastase, proteinase 3 (PR3) and cathepsin G, but unlike them is without proteolytic activity. To test whether, in addition to these proteases, azurocidin might be a target for the humoral autoimmune responses associated with human vasculitis, 185 indirect immunofluorescence (IIF)-positive ANCA sera, made up of four groups of sera with specificities for PR3 (n=37), myeloperoxidase (MPO; n=50), bactericidal/permeability-increasing protein (BPI; n=41) and sera that recognized none of them (triple negative, n=57), and 46 normal sera were screened for IgG anti-azurocidin antibodies using an ELISA incorporating purified azurocidin. Twenty of the 185 IIF-positive sera and 2/46 normal sera displayed reactivity with azurocidin. Positive sera could blot the 27-kD band by Western blot analysis. Further study of the 20 positive sera revealed that: (i) 10 also had autoreactivity for MPO, of which six additionally recognized lactoferrin; (ii) two had reactivity with BPI; (iii) the remaining eight sera were positive only for azurocidin. All 20 sera were from patients with systemic vasculitis, and four of the six sera with triple reactivity (for azurocidin, MPO and lactoferrin) were from patients with hydralazine-induced vasculitis. We concluded that: (i) azurocidin is a novel ANCA antigen; (ii) anti-azurocidin antibodies from a subgroup of patients might represent the consequence of a drug-induced multi-clone activation.

Topics: Adult; Aged; Amino Acid Sequence; Antibodies; Antibodies, Antineutrophil Cytoplasmic; Antigens; Antimicrobial Cationic Peptides; Autoantibodies; Blood Proteins; Carrier Proteins; Cations; Chromatography, Ion Exchange; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lactoferrin; Male; Middle Aged; Molecular Sequence Data; Vasculitis

To characterize the antigen specificity of circulating anti-neutrophil cytoplasmic antibodies (ANCAs) in inflammatory bowel disease (IBD).. Analysis of the prevalence of circulating ANCAs in patients with ulcerative colitis and Crohn's disease, by both non-specific methods (immunofluorescence against fixed neutrophil leukocytes) and specific antigen techniques (against purified neutrophil leukocyte constituents).. Indirect immunofluorescence against fixed polymorphonuclear leukocytes, and solid-phase enzyme-linked immunosorbent assay (ELISA) against neutrophil constituents (alpha-granules, elastase, myeloperoxidase, cathepsin g, lysozyme and lactoferrin).. Although results using immunofluorescence were typical of other studies (ulcerative colitis positive in 41%, Crohn's disease in 10%), ELISA studies showed antibody activity against neutrophil components in 69% of patients with ulcerative colitis and 39% of those with Crohn's disease. Antibodies in ulcerative colitis were commonly directed (in descending order) against lysozyme, cathepsin G, elastase, and lactoferrin, and in Crohn's disease against lysozyme.. Correlation of indirect immunofluorescence data and ELISA results indicated that even this large panel of specific antigens fails to identify all the ANCA targets in IBD. The lack of correlation between the findings of ANCAs, either in general or versus a specific target, and disease extent or activity in ulcerative colitis supports the suggestion that ANCAs are unlikely to be of primary importance in pathogenesis.

Topics: Adult; Aged; Antibodies; Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Cathepsin G; Cathepsins; Cholangitis, Sclerosing; Colitis, Ulcerative; Crohn Disease; Cytoplasmic Granules; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin G; Inflammatory Bowel Diseases; Lactoferrin; Leukocyte Elastase; Male; Middle Aged; Muramidase; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases; Vasculitis

We aimed at confirming the antigen specificity recognized by anti-neutrophil cytoplasm antibodies (ANCA) in patients presenting systemic vasculitis with anti-myeloperoxidase (MPO) activity on ELISA. Thirty-five consecutive patients with reactivity in anti-MPO ELISA and systemic microscopic vasculitides were tested in slot and Western blot analyses. Eleven of 35 sera exhibited binding in Western blot studies with the MPO preparation used in the ELISA: five sera bound at the size of MPO, but five sera reacted with a 78 kD species (p78) co-purifying with MPO, and one serum blotted both MPO and p78. Sequence analysis and antigen-specific assays including Western blot studies showed that p78 is lactoferrin. All anti-lactoferrin positive sera, but no anti-MPO positive sera, also exhibited anti-nuclear binding on HEp2 cells with specificity for histone. We concluded that: (a) a subgroup of patients presenting systemic vasculitis with false anti-MPO reactivity on ELISA had anti-lactoferrin antibodies; (b) anti-lactoferrin was associated with anti-nuclear activity with specificity for histone; (c) these patients had systemic vasculitis without histological evidence of immune complex deposition.

Topics: Amino Acid Sequence; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Autoantibodies; Biomarkers; Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Histones; Humans; Lactoferrin; Molecular Sequence Data; Peroxidase; Vasculitis

Antibodies directed against neutrophil granulocyte components have gained an increasing importance in diagnosing systemic vasculitis diseases. The present study was aimed to investigate distribution of anti-lactoferrin antibodies in systemic lupus erythematosus, the hydralazine-induced SLE-like syndrome, and in rheumatoid arthritis compared to RA complicated with vasculitis. Antibodies were detected by ELISA and verified by Western blotting and inhibition assay. Sera positive for IgM were absorbed to remove the rheumatoid factor. IgG and IgM anti-lactoferrin antibodies were found in SLE in 5% and 10% respectively. All patients with hydralazine-induced SLE had antibodies of both isotypes and the antibody level declined rapidly after withdrawal of the drug. In rheumatoid arthritis no IgG anti-lactoferrin antibodies were found, but 20% of the patients with vasculitis had IgM antibodies. Anti-lactoferrin antibodies seem partly to discriminate between genuine and hydralazine-induced SLE, which might indicate a pathogenic relevance in drug-induced autoimmunity. In uncomplicated rheumatoid arthritis it can be concluded that anti-lactoferrin antibodies lack clinical, as well as pathogenic relevance.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Anti-Idiotypic; Arthritis, Rheumatoid; Female; Humans; Hydralazine; Immunoglobulin G; Immunoglobulin M; Lactoferrin; Lupus Erythematosus, Systemic; Male; Middle Aged; Vasculitis

Thirty-five sera with binding greater than 20% in a myeloperoxidase (MPO, Calbiochem) ELISA were tested in Western blot analyses. 5/35 blotted MPO, but 5/35 blotted lactoferrin (LF) contaminating the commercial MPO preparation. All the anti-LF positive sera, but none of the anti-MPO positive sera, also exhibited anti-nuclear binding on Hep2 cells. Three of the patients with anti-LF antibodies had vasculitis affecting areas additional to the pulmonary-renal involvement which characterised the patients with anti-MPO antibodies.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Antibody Specificity; Autoantibodies; Blotting, Western; Cyanogen Bromide; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Lactoferrin; Lupus Erythematosus, Systemic; Peroxidase; Sequence Analysis; Sequence Homology; Vasculitis

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Blotting, Western; Cathepsin G; Cathepsins; Cytoplasmic Granules; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Lactoferrin; Muramidase; Myeloblastin; Neutrophils; Pancreatic Elastase; Peroxidase; Proteins; Serine Endopeptidases; Vasculitis

Anti-lactoferrin antibodies (anti-LF Ab) are more frequently found in patients with rheumatoid arthritis (RA) complicated by vasculitis when compared to patients with uncomplicated RA. Therefore the detection of anti-LF Ab in serum of patients with RA may be useful in the diagnosis of vasculitis in RA patients.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Lactoferrin; Vasculitis

In this study we examined the sera from 42 randomly selected patients with RA. ANCA was demonstrated by indirect immunofluorescence on human granulocytes in 10 patients with active disease and in none of the patients with inactive disease. ELISA's for the detection of specific antigens showed the presence of anti-myeloperoxidase in 3 patients, and anti-lactoferrin in 1 patient. The specificity of the remaining antibodies was unidentified. All 3 patients with antibodies to myeloperoxidase had vasculitis.

Topics: Antibodies, Antineutrophil Cytoplasmic; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Fluorescent Antibody Technique; Humans; Lactoferrin; Peroxidase; Vasculitis

To determine the occurrence of antineutrophil cytoplasmic antibodies (ANCA) and the specificity of these antibodies (Ab) in serum from patients with rheumatoid arthritis (RA) and patients with rheumatoid arthritis complicated by vasculitis (rheumatoid vasculitis [RV]).. ANCA was detected with an indirect immunofluorescence test on ethanol-fixed granulocytes. Ab against the cytoplasmic antigens proteinase-3, elastase, lactoferrin (LF), and myeloperoxidase were measured by enzyme-linked immunosorbent assay.. ANCA were found in the serum of 43% of 49 patients with RV and in 36% of 50 patients with RA. Anti-LF Ab occurred more frequently in RV patients (45%) than in RA patients (4%), whereas reactivity against the other cytoplasmic antigens did not differe significantly between these groups.. Anti-LF Ab in serum of patients with RA may be useful in the diagnosis of vasculitis in RA.

Topics: Antibodies; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Arthritis, Rheumatoid; Autoantibodies; Enzyme-Linked Immunosorbent Assay; Epitopes; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Lactoferrin; Vasculitis

Previously it was shown that tissue injury occurring in acute immune-complex-induced vasculitis, which is complement and neutrophil-dependent, is significantly attenuated by the presence of catalase, suggesting the pathogenic role of H2O2 generated from activated neutrophils. We now show that significant protection is also afforded by pretreatment of animals with apolactoferrin , a naturally occurring chelator of iron. Iron-saturated lactoferrin is devoid of protective effects. Deferoxamine mesylate, a synthetic iron chelator, also has protective effects. Infusion of ionic iron, especially Fe(III), potentiates the tissue injury. Significant protection from tissue injury is also produced by treatment of rats with dimethyl sulfoxide, a potent hydroxyl radical scavenger. Morphologically, animals treated with these protective interventions show the influx of neutrophils into sites of immune complex deposition, but there is markedly attenuated edema, little or no hemorrhage, and little evidence of endothelial cell injury, in contrast to the findings in nonprotected animals. These data support the suggestion that immune-complex-induced injury may be linked to generation of H2O2 from activated neutrophils and the subsequent conversion of H2O2 to the hydroxyl radical.

Topics: Animals; Apoproteins; Arthus Reaction; Chlorides; Deferoxamine; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Ferric Compounds; Ferrous Compounds; Free Radicals; Hydroxylation; Immune Complex Diseases; Lactoferrin; Male; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Specific Pathogen-Free Organisms; Vasculitis