IgA rheumatoid factor in primary Sjogren's syndrome.

OBJECTIVE: To assess the serum level of immunoglobulin A rheumatoid factor (IgA-RF) in patients with primary Sjogren's syndrome (pSS) and study the association with immunological and clinical factors. METHODS: Sera from 97 pSS patients diagnosed according to the preliminary European criteria and 100 controls were analysed for IgA-RF in a cross-sectional study design. RESULTS: IgA-RF was detected in serum of 25.8% of the pSS patients and in 1% of the controls. In patients with positive vs. negative IgA-RF, the focus scores in biopsy of the minor salivary glands were 4.41 and 1.43 (p<0.0001), respectively. There was a correlation between positive IgA-RF and positive antinuclear antibodies (ANA) (r = 0.263, p<0.009), IgM-RF (r = 0.70, p<0.0001), anti-SSA/SSB (r = 0.73, p<0.0001), and a high serum level of IgG (r = 0.59, p<0.0001). The presence of renal disease was higher in IgA-RF-positive vs. negative pSS patients (20.0% vs. 5.6%, p = 0.047). The serum level of the hormone prolactin (PRL) correlated to the serum level of IgA-RF (r = 0.31, p = 0.026). CONCLUSIONS: The presence of IgA-RF in patients with pSS is closely associated with the presence of autoantibodies, and with focus scoring in biopsies of the salivary glands. IgA-RF is associated with renal disease in pSS but we found no correlation to other extraglandular manifestations.

Incidence of thromboembolic events in patients with primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a connective tissue disease with symptoms and serological findings often overlapping with systemic lupus erythematosus (SLE) (1). Thromboembolic events are common in SLE but not in pSS (2)(3). However, case reports have described pSS patients who developed fulminant multiorgan disease due to thrombotic diathesis 4, and we have presented a case with acute catastrophic anti-phospholipid syndrome (APS) in a pSS patient (5). In this study we wanted to examine the incidence of thromboembolic episodes and relate these to the presence of autoantibodies and coagulation abnormalities in 90 pSS patients during a 4.6-year follow-up.

Estimation of the prevalence of primary Sjögren's syndrome in two age-different community-based populations using two sets of classification criteria: the Hordaland Health Study.

OBJECTIVE: To estimate the point prevalence of primary Sjögren's syndrome (pSS) in two populations, aged 40-44 and 71-74 years, using two sets of classification criteria. METHODS: The participating individuals were recruited from the Hordaland Health Study (HUSK) conducted during 1997-99. A total of 18 592 individuals born 1953-57 and 3346 individuals born 1925-27 were sent a questionnaire covering various health-related questions, including four questions about sicca symptoms. Among those answering positive to at least one of the four questions, 99 and 90 individuals born 1953-57 and 1925-27, respectively, were examined further. For diagnosis of pSS two classifications were used, the preliminary European criteria from 1993, and the revised European criteria from 1996. RESULTS: By using the two classification criteria from 1993 and 1996, the point prevalences were 0.44% [95% confidence interval (CI) 0.34-0.57] and 0.22% (95% CI 0.15-0.32), respectively, for the population group born 1953-57. The corresponding estimates were 3.39% (95% CI 2.77-4.14) and 1.40% (95% CI 1.02-1.92) for the population born 1925-27. CONCLUSION: The point prevalence of pSS was approximately seven times higher in the elderly population aged 71-74 years compared to individuals aged 40-44 years, regardless of the classification criteria used.

A study of the arthritis pattern in primary Sjögren's syndrome.

OBJECTIVE: To study the frequency and pattern of arthritis in primary Sjögren's syndrome (pSS), and its association with clinical and immunological factors. METHODS: 102 patients with pSS diagnosed according to the preliminary European Classification Criteria were examined yearly for 4.5 years in a prospective study design. Arthralgia and arthritis were registered during the 459 patient-years observation period. RESULTS: Arthralgia was reported by 75 patients (73.5%) and arthritis was demonstrated in 18 patients (17.6%) during the observation period. The most commonly affected joints were ancles (n = 7), MCP joints (n = 6), shoulders (n = 6), MTP joints (n = 6) and wrists (n = 5). Symmetrical bilateral arthritis were most commonly observed in ancles (4 patients) and wrists, shoulders and MTP joints. Five patients had longstanding arthritis observed at more than one clinical examination, and one developed seronegative rheumatoid arthritis. Arthralgia/arthritis was not correlated to any clinical or immunological factors, and usually ESR and CRP were normal when arthritis was observed. CONCLUSION: Arthritis in pSS is usually mild, resolving, and unrelated to other clinical and immunological factors. A typical pattern is uni- and bilateral arthritis in the ankles, but joints in hands, feet and shoulders may also be affected.

Amphipathic variable region heavy chain peptides derived from monoclonal human Wegener's anti-PR3 antibodies stimulate lymphocytes from patients with Wegener's granulomatosis and microscopic polyangiitis.

Amphipathic variable-region heavy chain 11-mer peptides from monoclonal human IgM antiproteinase-3 antibodies were studied for peripheral blood lymphocyte stimulation in 21 patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), connective tissue disease controls and normal control subjects. Positive T-cell activation was observed in most experiments with WG patients' lymphocytes using amphipathic VH-region peptides from four different human monoclonal anti-PR3 antibodies. Control peptides of the same length but without amphipathic characteristics along with other amphipathic peptides not derived from monoclonal anti-PR3 sequence were employed as controls. No significant lymphocyte stimulation was observed with normal controls, but positive stimulation with amphipathic VH peptides was also recorded in other connective tissue disease controls mainly patients with rheumatoid arthritis. Amphipathic peptides not derived from anti-PR3 sequence did not stimulate WG lymphocytes. Our findings indicate that lymphocyte reactivity as an element of cell-mediated immunity may be activated by amphipathic VH-region amino acid sequences of autoantibodies which are themselves associated with diseases such as WG.

Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function.

We recently demonstrated that intravenous (i.v.) injection of the iron-binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron-saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood-tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (Pif) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB-albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron-free or iron-saturated Lf (both 100 microg mL-1) in the absence and presence of 0.5 mM hydrogen peroxide. Pif increased significantly at 11-30 min following Lf to +2.1 +/- 0.3 and +1.7 +/- 0.2 mmHg at 11-20 and 21-30 min, respectively, compared with +0.1 +/- 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB-albumin during 3 h was not affected by iron-free or iron-saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf-induced increase in albumin extravasation in rat skin is not explained by changes in Pif (because Lf raised Pif significantly) or direct effects of Lf on the endothelial barrier.

What you should know about PR3-ANCA. Structural aspects of antibodies to proteinase 3 (PR3).

Reactive antigenic epitopes on presumed autoantigens of biologic interest have been examined by many researchers. The central third complementarity-determining region (CDR3) residues of a human monoclonal anti-proteinase 3 (PR3) antibody contained many negatively charged aspartic acid residues, perhaps contributing to its reactivity with positively charged PR3 regions. Examination of four other human monoclonal anti-PR3 antibodies shows a number of negatively charged residues within their CDR3 regions. Mapping of segments of linear PR3-epitopes reacting with anti-neutrophil cytoplasmic antibodies (ANCA) demonstrated a preliminary estimate of structures contributing to antigenic determinants. T-cell epitopes on PR3 are reported in studies of chronic myeloid leukemia. These T-cell epitopes appear to be human leukocyte antigen (HLA) A2.1 restricted.

Lactoferrin and anti-lactoferrin antibodies: effects of ironloading of lactoferrin on albumin extravasation in different tissues in rats.

Lactoferrin is a cationic iron-binding protein, which is released from activated neutrophils in concert with reactive oxygen species. In vitro, lactoferrin has both anti- and proinflammatory effects; many of them dependent on iron-binding. In vivo, only iron-free lactoferrin reduced inflammatory hyperpermeability in the lung. We therefore examined whether 1 mg iron-free (Apo-Lf) or iron-saturated lactoferrin (Holo-Lf) alone or followed by anti-lactoferrin antibodies (aLf) affected permeability evaluated by extravasation of radiolabelled bovine serum albumin (CBSA) in different tissues of anaesthetized rats. Fifteen minutes after i.v. injection of Lf, aLf or saline was given and circulatory arrest was induced 20 min thereafter. Measurements were performed in control, after Apo-Lf, Holo-Lf, Apo-Lf + aLf, Holo-Lf + aLf and aLf alone (n=6-8 in each group). No intergroup differences were found for plasma volume and haematocrit at the start and end of the 37 min extravasation period or for total tissue water in any of the six different tissues studied, excluding larger transcapillary fluid shifts. However, increases in CBSA were seen without differences in tissue intravascular volume. Iron-free lactoferrin and aLf alone did not change CBSA significantly. Iron-saturated lactoferrin significantly increased CBSA in skin (neck), trachea and left ventricle of the heart to 249 +/- 9, 284 +/- 16 and 160 +/- 7% of control, respectively. When followed by aLf, both Apo- and Holo-Lf increased CBSA significantly in four and five of the tissues studied, respectively. However, no significant effect was seen for Holo-Lf + aLf compared with Holo-Lf alone. In conclusion, iron-saturated, but not iron-free lactoferrin increased CBSA, whereas antilactoferrin increased CBSA compared with lactoferrin alone only when following iron-free lactoferrin.

Apoptosis and cell penetration by autoantibody may represent linked processes.

An hypothesis is presented in which the natural physiologic process of apoptosis may sometimes be linked to cell penetration by autoantibody. Apoptotic blebs as residuals of the programmed cell death process have recently been demonstrated to contain clusters of disparate autoantigens which have previously been implicated in the generation of autoimmune reactions. In parallel, there now exists a mounting body of evidence that certain autoantibody molecules are capable of living cell penetration and even nuclear membrane ingress. We suggest that these two processes, apoptosis and cell penetration by autoantibody, may be associated with one another in certain circumstances.

Transportation and storage of cryosections in PBS-Dulbecco.

Tissue pieces that cannot be fixed and embedded before immunostaining must be cryosectioned and stained quickly. We describe an easy and reliable technique to keep cryosections with immune complexes and complement stored for several weeks until the final immunostaining. The same technique was used for long distance transportation of cryosections and it was extensively tested for human kidney biopsies with excellent results.

Determination of primary amino acid sequence and unique three-dimensional structure of WGH1, a monoclonal human IgM antibody with anti-PR3 specificity.

Transformed B cells making monoclonal IgM-lambda anti-PR3 antibody WGH1 from a patient with Wegener's granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human micro and lambda chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH regions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH sequence was subgroup III with marked overall homology to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been determined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VHCDR3 contains a loop which shows a major projection not usually recorded in other previously studied antibody molecules.

Hepatic and extrahepatic clearance of circulating human lactoferrin: an experimental study in rat.

Lactoferrin, unlabelled or 125I-labelled by 2 different methods, was given intravenously to rats. Blood, tissue and liver cell radioactivity was measured. Both of the radiolabelled preparations were eliminated by the liver, and some deposited extrahepatically. One preparation formed large aggregates--here 90% of the hepatic uptake occurred in the Kupffer cells. The other preparation, consisting mostly of protein monomers but also dimers/oligomers/microaggregates, was taken up by hepatocytes (63% of total liver uptake), liver endothelial cells (22%) and Kupffer cells (15%). On a per cell volume basis, lactoferrin uptake was much more efficient by nonparenchymal cells compared to hepatocytes, which explains why immunomorphological staining only revealed lactoferrin in the nonparenchymal liver cells. The study demonstrates that radio-iodination of lactoferrin can affect its properties and handling, which may be important regarding contradictory reports on hepatic lactoferrin uptake. We conclude that both hepatocytes and nonparenchymal liver cells are involved in the blood clearance of lactoferrin, probably to a great extent owing to nonspecific mechanisms. Extrahepatic deposition and exposure (for instance on vessel walls/glomeruli) suggests that lactoferrin can be available to circulating anti-lactoferrin autoantibodies in autoimmune disease.

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren's syndrome.

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sjögren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren's syndrome.

Serum levels of adhesion molecules and cytokines in patients with acute leukaemia.

The cytokine network and the adhesion molecule system are intercellular signal pathways. The cytokine effects are modulated in vivo by soluble cytokine antagonists, whereas the cell to cell contact mediated by adhesion molecules and their ligands may be blocked by the soluble forms of the adhesion molecules. The cytokine network is important for proliferation and cytokine secretion by acute leukaemia blasts, and membrane-bound adhesion molecules are important for blast interactions with neighbouring cells of the in vivo microenvironment. Both these signal systems are operative during the period of cytopenia following intensive chemotherapy for acute leukaemia. In the present review, we discuss the influence of disease status, chemotherapy and complicating infections on serum levels of cytokines and soluble adhesion molecules in acute leukaemia patients. We have demonstrated increased serum levels of both cytokines and cytokine antagonists in acute leukaemia patients with complicating bacterial infections during chemotherapy-induced cytopenia. Serum levels of the selectin adhesion molecules were decreased during bacterial infections in leukopenic patients compared to healthy individuals. In contrast, the intercellular adhesion molecule-1 response and the cytokine/cytokine antagonist responses were qualitatively similar to responses seen in previously healthy individuals with serious bacterial infections.

Leucocyte activation by anti-lactoferrin antibodies bound to vascular endothelium.

Human polymorphonuclear neutrophil leucocytes (PMNL) prestimulated with the formylated tripeptide f-Met-Leu-Phe (fMLP) were activated to an immediate chemiluminescence (CL) response by polyclonal rabbit antibodies against human lactoferrin (Lf). This activation, indicating the formation of reactive oxygen species, was induced by intact IgG antibodies but could not be brought about by F(ab')2 fragments. Human Lf was also shown to adhere to the surface of cultured bovine aorta endothelial cells (BEC). When Lf-coated BEC grown on microcarrier beads were reacted with anti-Lf antibodies, an immediate CL response was achieved also with nonprimed PMNL. Here, too, the reaction required intact IgG antibodies. Also, patient sera containing anti-Lf autoantibodies of IgG class were shown to activate fMLP-treated PMNL. The same effect was obtained (in a dose-dependent manner) with the gammaglobulin fraction from anti-Lf-positive serum. Further, anti-Lf-antibody-positive patient sera incubated with Lf-coated BEC beads were also able to activate non-stimulated PMNL to a chemiluminescence response. The results are discussed in relation to possible mechanisms of cell/tissue damage induced by anti-neutrophil cytoplasmic antibodies (ANCA).

Distribution of lactoferrin and 60/65 kDa heat shock protein in normal and inflamed human intestine and liver.

Immunisation against the mycobacterial heat shock protein (hsp-65) has been proposed to lead to production of autoantibodies against human lactoferrin. Such antibodies occur in ulcerative colitis and in primary sclerosing cholangitis. This study analysed the distribution of hsp-65 and lactoferrin in biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis and studied whether immunisation against mycobacterial hsp-65 resulted in production of antilactoferrin antibodies and vice versa. Polyclonal rabbit antihuman lactoferrin and monoclonal mouse anti-hsp-65 (ML30) were used for immunohistochemistry on biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis. Rats were immunised against human lactoferrin and mycobacterial hsp-65 respectively. Antibody measurements were done by enzyme immunosorbent assays. It was found that lactoferrin and hsp-60/65 were not codistributed. Lactoferrin was found on vascular endothelium and in nonparenchymal liver cells both in inflamed and uninflamed tissues, but only in the hepatocytes of inflamed liver. ML30 reactivity was not inhibited by antilactoferrin antibodies. Rat anti-hsp-65 serum had no detectable antilactoferrin antibodies. In conclusion, antilactoferrin antibodies probably do not arise by immunisation against mycobacterial hsp-65. Both nonparenchymal cells and hepatocytes probably participate in clearance of lactoferrin. Endothelial exposure of lactoferrin may have pathogenic implications in diseases with antilactoferrin autoantibodies.

Antineutrophil cytoplasmic antibodies in reactive arthritis.

OBJECTIVE: To study the occurrence of antineutrophil cytoplasmic antibodies (ANCA) in reactive arthritis (ReA). METHODS: Sera from 22 patients with ReA were analyzed by ELISA for the presence of autoantibodies (IgG and IgA) against a proteinase-3 containing azurophilic granule extract ("alpha-antigen") from human polymorphonuclear leukocytes, myeloperoxidase (MPO), and lactoferrin (Lf), respectively. Rheumatoid factor (RF), antinuclear antibodies (ANA), and HLA-B27 were also tested. Erythrocyte sedimentation rate and serum levels of C-reactive protein were used to assess disease activity. The patients were divided into acute or chronic (> 1 year) disease. RESULTS: 12/22 patients (55%) had IgG ANCA (7 had MPO ANCA, 8 had Lf ANCA, and 4 had alpha-ANCA). Eight patients (36%) had IgA ANCA. One serum was positive only for IgA ANCA. 18/21 patients (86%) were HLA-B27 positive, and none had RF or ANA. The triggering infection was Chlamydia trachomatis in 6 cases. Campylobacter jejuni in 6, Yersinia enterocolitica in 4. In 6 patients the causative microorganism could not be determined. ANCA was more prevalent in chronic disease (6/7, 82%) compared to acute (7/15, 47%). No obvious correlation was seen between ANCA and disease activity. CONCLUSION: ANCA, predominantly those reacting with Lf and/or MPO preparations, are common in ReA.

Perinuclear anti-neutrophil cytoplasmic antibody and refractory pouchitis. A case-control study.

Refractory pouchitis (RP) is a debilitating complication of ileal pouch reservoirs that affects approximately 2.5% of patients. Although the cause of RP is unknown, it is frequently hypothesized that it reflects underlying Crohn's disease. Since perinuclear anti-neutrophil cytoplasmic antibody (pANCA) is found in approximately 70% of ulcerative colitis patients but only rarely in Crohn's disease patients, it may help distinguish Crohn's disease from ulcerative colitis. Therefore, to test whether RP reflects "missed" Crohn's disease, we determined the ANCA status of 26 patients with RP. The pANCA was positive in 42% of cases [50% of Kock pouch cases and 33% of ileoanal pull-through (IAPT) cases] and 57% of matched control subjects without pouchitis (N = 42, P = NS). Moreover, 3/6 (50%) of IAPT RP subjects whose signs and symptoms most suggested Crohn's disease tested positive for pANCA. When compared to controls, IAPT cases exhibited significantly more preoperative extraintestinal manifestations (EIMs) of inflammatory bowel disease (P < 0.05). The presence of preoperative EIMs was 100% predictive of postoperative EIMs (P < 0.05). Review of pouch biopsies from cases of RP revealed no pathognomonic histologic features of Crohn's disease. These data confirm our previous suggestion that RP does not reflect underlying Crohn's disease but may be associated with the EIMs of inflammatory bowel disease.