Faculty Bibliography

Neutrophils express receptors for numerous phlogistons which, when occupied, trigger distinct signal-transduction pathways. Previous studies have shown that stimulation of neutrophils with chemoattractants induces shedding of the adhesive molecule L-selectin and increased expression of the beta 2-integrin CD11b/CD18. We determined the effect of ligation of classic, G-protein-linked chemoattractant receptors [C5a, interleukin-8 (IL-8), formylmethionyl-leucylphenylalanine (FMLP) and substance P], receptors for the Fc portion of IgG (Fc gamma receptors) and receptors for transforming growth factor beta (TGF beta) on expression of adhesive molecules by neutrophils and the stimulus-transduction mechanisms thought to mediate these changes. We were surprised to observe that occupancy of Fc gamma receptors by immunocomplexes (BSA-anti-BSA) stimulated increased expression by neutrophils of CD11b/CD18 at concentrations which did not affect L-selectin expression (EC50 9 micrograms/ml versus 350 micrograms/ml respectively, P < 0.00001, n = 5). In contrast, similar to previous studies, recombinant C5a, recombinant IL-8 and FMLP all stimulated increased expression of CD11b/CD18 (170-260% of basal, P < 0.001, n = 5) and shedding of L-selectin (56-75% reduction from basal, P < 0.001, n = 5) at similar concentrations and with similar potencies (EC50 = 2, 5, and 3 nM respectively). In contrast, neither TGF beta 1 nor, surprisingly, substance P affected expression of CD11b/CD18 or L-selectin. The regulation of expression of CD11b/CD18 or L-selectin in response to FMLP or immunocomplexes was unaffected by cytochalasin B (5 micrograms/ml) or the tyrosine kinase inhibitor tyrphostin-25 (25 microM). Although occupancy of both chemoattractant (FMLP) and Fc gamma receptors stimulated increments in the second messenger diacylglycerol, disruption of actin microfilaments by cytochalasin B enhanced diacylglycerol generation in response to FMLP but not in response to ligation of Fc gamma receptors. Moreover, both FMLP and immune aggregates provoked fluxes of intracellular Ca2+ concentration which differed with respect to both magnitude and kinetics and did not correlate well with regulation of adhesive-molecule expression. As upregulation of CD11b/CD18 is tightly linked to exocytosis of specific granules, these results suggest that shedding of L-selectin by activated neutrophils is not linked to exocytosis. These studies provide further evidence that receptors for chemoattractants and immunocomplexes on the neutrophil are linked to multiple signalling pathways

The role of phosphorylation and dephosphorylation events in homotypic neutrophil aggregation mediated by CD11b/CD18 molecules was investigated using okadaic acid, an inhibitor of serine and threonine phosphatases. In the absence of exogenous stimuli the addition of okadaic acid to neutrophils resulted in a dose-dependent increase in phosphorylation of the CD18 beta chain that was further augmented by PMA but unaffected by FMLP. Phosphorylation induced by okadaic acid was reversed by staurosporine and minimally decreased by the less selective PKA/PKC inhibitors, H-7 and H-8. This suggests the existence of constitutive phosphatase and kinase activity emphasizing the dynamic state of phosphorylation and dephosphorylation of the beta 2 integrins. Unlike PMA, okadaic acid did not promote homotypic neutrophil aggregation. Furthermore, both the PMA-induced pathway of irreversible aggregation, blocked by staurosporine, as well as the FMLP-induced pathway of reversible aggregation, augmented by staurosporine, were inhibited by okadaic acid in a dose- and time-dependent manner. These results provide evidence that a phosphatase-dependent step is involved in each of these two distinct pathways that regulate neutrophil aggregation mediated by beta 2 integrin activation

OBJECTIVE: To determine the initial clinical status and the long-term outcome of mothers and their children with autoantibody-associated congenital heart block. DESIGN: Dynamic, longitudinal cohort study. PATIENTS: 55 children with isolated congenital heart block, their 52 mothers, and 5 other women currently carrying fetuses with congenital heart block. All maternal sera contained antibodies to SSA/Ro alone or to both SSA/Ro and SSB/La. MEASUREMENTS: Clinical information obtained from mailed questionnaires, telephone interviews, primary physicians, and chart reviews. RESULTS: When congenital heart block was identified in the children, 23 women were asymptomatic, 15 had systemic lupus erythematosus, 8 had the Sjogren syndrome, and 11 had an undifferentiated autoimmune syndrome. Follow-up ranged from 1 week to 20 years (median, 3.7 years). Eleven (48%) of the 23 initially asymptomatic mothers developed symptoms of a rheumatic disease (0.15 status changes/patient-year of follow-up; 6 (26%) developed an undifferentiated autoimmune syndrome, 2 (9%) developed the Sjogren syndrome, and 3 (13%) developed systemic lupus erythematosus. One mother with the Sjogren syndrome progressed to systemic lupus erythematosus. Four (16%) of 25 subsequent pregnancies in 22 women were complicated by heart block. Seventeen affected children died, 12 within 1 month of birth. Pacemakers were implanted in 37 (67%) of the 55 children, 27 within 3 months after birth. CONCLUSION: The development of rheumatic disease in asymptomatic mothers identified by the birth of a child with congenital heart block is common but not universal. The risk for congenital heart block in subsequent pregnancies is low. One third of the children with autoantibody-associated congenital heart block die in the early neonatal period and, of those who survive, most require pacemakers

Abs to the 52-kDa SS-A/Ro protein are found in high prevalence in patients with Sjogren's syndrome (SS) and mothers whose children have the neonatal lupus syndrome (NLS). This study further defines the specificity of this response. By ELISA, 97% of 59 mothers of offspring with NLS had Abs to the 52-kDa recombinant protein compared with 80% in 132 non-NLS sera with anti-SS-A/Ro Abs (p 0.004). Antigenic regions on the 52-kDa protein were evaluated by immunoprecipitation of [35S]-radiolabeled in vitro translation products. Ninety-five percent of 99 sera that contained anti-52-kDa Abs by ELISA reacted with a large fragment spanning amino acids (aa) 1-291. Two antigenic regions were identified, aa169-291 containing the leucine zipper that was recognized by 83% of the anti-52-kDa sera tested and aa1-78 containing the zinc finger domains that was recognized by only half the sera. No sera immunoprecipitated this N-terminal fragment exclusively. Recognition of one or both regions was not unique to any clinical subset of patients; however, a greater number of sera from patients with SS contained both specificities, whereas asymptomatic mothers whose children had NLS comprised the only clinical group in which the majority recognized the central region of the molecule. Reactivity with both epitopes was demonstrated significantly more often in sera with high titers of Abs to the 60-kDa rSS-A/Ro protein by ELISA in association with the anti-52-kDa response compared with anti-52-kDa responses associated with low titers of anti-60-kDa Abs (p < 0.04). Eighty-one percent of 16 sera that recognized the N-terminal epitope were from patients with the combination of HLA-DRB1*0301, DQA1*0501, and DQB1*0201 alleles, compared with 30% of 10 that recognized only the central epitope (p < 0.02). In summary, this study demonstrates that there are at least two antigenic determinants on the 52-kDa SS-A/Ro protein, one 'immunodominant' and the other recognized by a more 'restricted' subset of anti-52-kDa SS-A/Ro Abs

OBJECTIVE. To test the hypothesis that during exacerbations of systemic lupus erythematosus (SLE), endothelial cells are activated to increase their expression of adhesion molecules. METHODS. Endothelial cell expression of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) was quantitated immunohistochemically in 20 biopsy specimens from nonlesional, non-sun-exposed skin from 16 SLE patients. Disease activity was evaluated with the SLE Disease Activity Index (SLEDAI) and with measurements of complement components C3a desArg, C3, and C4. RESULTS. The mean expression of all 3 adhesion molecules was significantly elevated in patients with SLE versus healthy controls, as well as in patients with active versus inactive SLE. The mean C3a desArg level was significantly higher in patients with active SLE compared with those with inactive SLE. The SLEDAI scores correlated directly with C3a desArg levels and inversely with C3 and with C4 levels. Evaluation of serial biopsy specimens demonstrated loss of endothelial cell adhesion molecules and reduction of C3a levels with clinical improvement. CONCLUSION. Our findings demonstrate up-regulation of the surface expression of 3 distinct adhesion molecules, E-selectin, VCAM-1, and ICAM-1, in patients with SLE. The abnormal expression of these endothelial cell adhesion molecules is most marked in patients with active disease characterized by significant elevations of the complement split product C3a desArg. We suggest that in certain SLE patients, excessive complement activation in association with primed endothelial cells induces leukocyte-endothelial cell adhesion and leuko-occlusive vasculopathy

Liver disease in the fetus and neonate may be associated with maternal Sjogren's syndrome (neonatal lupus erythematosus). The infant of a mother with Sjogren's syndrome and high anti-Ro/SS-A and anti-La/SS-B antibody titers presented at 2 1/2 weeks of age with overwhelming hepatic insufficiency and died at 6 weeks of age. Autopsy confirmed the antemortem diagnosis of neonatal hemochromatosis. We discuss the possibility of a relationship between these two conditions

OBJECTIVE. To identify the fine specificity patterns of maternal anti-SS-A/Ro and anti-SS-B/La antibodies that are associated with the birth of a child with transient or permanent manifestations of neonatal lupus syndromes, and to suggest a predictor algorithm for use in counseling. METHODS. Sera were obtained from 4 groups of mothers: 57 whose children had congenital heart block, 12 whose children had transient dermatologic or hepatic manifestations of neonatal lupus but no detectable cardiac involvement, 152 with systemic lupus erythematosus and related autoimmune diseases, who gave birth to healthy infants, and 30 with autoimmune diseases whose pregnancy resulted in miscarriage, fetal death, or early postpartum death unrelated to neonatal lupus. Antibodies to SS-A/Ro and SS-B/La were assessed by enzyme-linked immunosorbent assay (ELISA) and by sodium dodecyl sulfate (SDS)-immunoblot. RESULTS. Anti-SS-A/Ro antibodies were identified by ELISA in 100%, 91%, 47%, and 43% of the mothers of infants with heart block, with transient neonatal lupus, healthy infants, and fetal death, respectively. High titers of anti-SS-A/Ro antibodies were present more often in mothers of children with cardiac disease or transient neonatal lupus than in either of the other 2 groups. Maternal antibodies to SS-B/La were detected by ELISA in 76% of the heart block group, 73% of the cutaneous neonatal lupus group, 15% of the group with healthy children, and 7% of the fetal death group. On SDS-immunoblot, sera from 91% of the heart block group mothers who had antibodies to SS-A/Ro but not to SS-B/La recognized at least 1 SS-A/Ro antigen, with significantly greater reactivity against the 52-kd component. In contrast, only 62% of the anti-SS-A/Ro positive, anti-SS-B/La negative responders in the healthy group recognized the 52-kd and/or the 60-kd component. Although there was no profile of anti-SS-A/Ro response unique to the mothers of children with heart block or cutaneous manifestations of neonatal lupus, only 1% of the healthy infants were born to mothers with antibodies directed to both the 52-kd SS-A/Ro and 48-kd SS-B/La antigens and not to the 60-kd SS-A/Ro antigen. CONCLUSION. Women with antibodies to both SS-A/Ro and SS-B/La have an increased risk of giving birth to children with neonatal lupus, especially if the anti-SS-A/Ro response identifies the 52-kd component on SDS-immunoblot. Women whose sera contain only anti-SS-A/Ro antibodies in low titer and only recognize determinants that are altered by conditions of SDS-immunoblot have a low risk for giving birth to a child with neonatal lupus. Specific antibody profiles do not distinguish among the manifestations of the neonatal lupus syndromes