Wegener granulomatosis.
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Antibodies, Antineutrophil Cytoplasmic↗
Biomedical subjects
M A Arnaout
Publications and source records attributed to M A Arnaout.
Genetic cause of leukocyte adhesion molecule deficiency. Abnormal splicing and a missense mutation in a conserved region of CD18 impair cell surface expression of beta 2 integrins.
Patients with leukocyte adhesion molecule (CD11/CD18, beta 2 integrins) deficiency have structural defects in the common beta subunit (CD18), which prevent heterodimer formation and normal cell surface expression of these receptors, leading to life-threatening bacterial infections. To elucidate the nature of these defects in a patient with partial (type II) deficiency, abnormal CD18 cDNA clones were isolated, using the polymerase chain reaction to amplify the patient's B cell-derived cDNAs. Sequence analysis revealed two mutant alleles. cDNA clones, representing a maternal allele, contained both a 12-base pair insertion resulting in an in-frame addition of four amino acids between P247 and E248 and a C1756----T nucleotide transition, resulting in an R586----W substitution in the normal CD18 protein. The inframe insertion arose by a single nucleotide C----A transversion in the 3' terminus of an intron, generating aberrant splice acceptor site. Other cDNA clones contained an A1052----G nucleotide transition not present in either parent which resulted in an N351----S substitution. To determine the functional importance of these changes, cDNA encoding a normal alpha chain (CD11b) was cotransfected into COS with CD18 cDNAs encoding for wild-type, maternal mutant allele, or CD18 containing N351----S substitution. Immunostaining of transfectants with anti-CD18 monoclonal antibodies revealed no cell surface expression of the maternal mutant CD18, and 22% surface expression of N351----S CD18. Both the insertion and the N351----S mutations occurred in a 250 amino acid extracellular region of CD18 that is highly conserved among beta integrins supporting a role for this region in heterodimer formation.
Amino Acid Sequence↗
Antineutrophil cytoplasmic autoantibody-associated vasculitis presenting as Sjögren's syndrome.
A 63-year-old woman, in whom a diagnosis of Sjögren's syndrome was initially made, proved to have systemic vasculitis with salivary gland involvement and necrotizing and crescentic glomerulonephritis. Antineutrophil cytoplasmic autoantibodies (ANCA) against myeloperoxidase were positive. ANCA-associated vasculitis should be considered in the differential diagnosis of Sjögren's syndrome. A positive finding on immunoassay for ANCA against myeloperoxidase or proteinase 3 may help establish the diagnosis.
Antibodies, Antineutrophil Cytoplasmic↗
On the regulation of beta 2 integrins.
The complex functions played by beta 2 integrins in mediating a large variety of adhesive interactions of leukocytes are highly regulated. This regulation results in transient adaptations/associations, permitting physical and functional recycling of these receptors during chemotaxis, phagocytosis and target-cell killing. The structural definition of these adaptations will lead not only to a better understanding of how these receptors are regulated in leukocytes but also shed valuable light on how these integrins integrate diverse extracellular signals into spatially and temporaly coordinated cellular responses.
Amino Acid Sequence↗
Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways.
Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.
Cell Movement↗
Late-onset congenital adrenal hyperplasia in women with hirsutism.
Hirsutism in women is a clinical manifestation of excessive production of androgens. The source of the excess androgen may be either the ovaries or the adrenal glands, or increased peripheral conversion of weak androgenic hormones to more potent androgens. Late-onset (non-classic) congenital adrenal hyperplasia is a cause of hirsutism in adult women, but its frequency and the patterns of abnormalities in adrenal hormone secretion are not well understood. The frequency of non-classical adrenal hyperplasia due to deficiencies of 3 beta-hydroxy-delta 5-steroid dehydrogenase, 21-hydroxylase, and 11 beta-hydroxylase among 65 women with hirsutism were determined. All enzyme defects were identified by comparing the patients' hormonal responses to 0.25 mg intravenous bolus of alpha 1-24-ACTH with those of age-matched normal women. The hormones measured in plasma during the ACTH stimulation tests were progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, DHEA-sulfate, androstenedione, testosterone, 11-deoxycortisol, and cortisol. Similarly these hormones were measured after overnight 1 mg oral dexamethasone. Twelve women (18.5%) had 3 beta-hydroxy-delta 5-steroid dehydrogenase deficiency, 24 (37%) 21-hydroxylase deficiency, and 14 (21.5%) 11 beta-hydroxylase deficiency. Women with 21-hydroxylase deficiency also had evidence of a partial deficiency in 11 beta-hydroxylase activity (12 of the 24 patients). Similarly, most (11 of the 14) of the women with 11 beta-hydroxylase deficiency also had evidence of a deficiency in 3 beta-hydroxy-delta 5-steroid dehydrogenase activity. Among the 15 patients with no adrenal biosynthetic defect, eight had high plasma androgen concentrations, and seven had normal concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Hydroxysteroid Dehydrogenases↗
Effect of an anti-Mo1 MAb on ozone-induced airway inflammation and airway hyperresponsiveness in dogs.
Ozone inhalation causes neutrophil migration into the airway and airway hyperresponsiveness in dogs. The leukocyte adhesion molecule Mo1 (CD11b/CD18) is a heterodimeric glycoprotein the expression of which is necessary for neutrophil adhesion to endothelium. To evaluate the contribution of Mo1 to ozone-induced neutrophil influx and airway hyperresponsiveness, six dogs were treated intravenously with an Anti-Mo1 monoclonal antibody (3.75 mg/kg in normal saline) that binds to both human and canine Mo1, or the diluent alone, 1.5 h before inhaling ozone (3 ppm for 30 min), or dry air. Airway responses to doubling doses of inhaled acetylcholine (ACh) were measured before and after inhalation of ozone. Neutrophil influx was assessed by bronchoalveolar lavage (BAL) performed after the second ACh inhalation. Treatment with anti-Mo1 prevented the ozone-induced influx of neutrophils into BAL. After diluent and inhaled dry air, the neutrophil count in BAL was 1.49 +/- 1.26 (SE) x 10(4) (5.0% of total cells). After diluent and inhaled ozone, the neutrophil count increased to 7.27 +/- 3.22 (SE) x 10(4) (22.6% of total cells) (P < 0.05). After anti-Mo1 and inhaled ozone, the neutrophil count was 1.48 +/- 0.62 (SE) x 10(4) (8.5% of total cells). Treatment with anti-Mo1 also significantly reduced the number of eosinophils in BAL after ozone. Ozone-induced ACh airway hyperresponsiveness was not prevented by treatment with anti-Mo1. These results indicate that expression of Mo1 is necessary for ozone-induced neutrophil migration into the airway lumen.
Acetylcholine↗
Arginine-vasopressin and endothelium-associated proteins in thyroid disease.
Plasma concentrations of endothelium-derived proteins (fibronectin and von Willebrand factor), liver synthesized proteins (haptoglobin, transferrin, ceruloplasmin, alpha 1-antitrypsin, antithrombin III and factor VIII-coagulant) and plasma arginine-vasopressin (AVP) were measured in 12 hyperthyroid, 9 hypothyroid and 15 age- and sex-matched normal controls. In hyperthyroid patients the plasma concentrations of AVP and endothelium-associated proteins (EAP) were significantly higher than in the control group (p less than 0.05 and p less than 0.01 respectively). Rendering hyperthyroid patients into the euthyroid state significantly lowered AVP (p less than 0.01), fibronectin (p less than 0.05) and von Willebrand factor (p less than 0.01) compared with pretreatment levels. Hypothyroid patients were studied at diagnosis and after replacement therapy with levothyroxine. Compared with pretreatment values, significant increases were noted in plasma concentrations of von Willebrand factor, fibronectin and AVP (p less than 0.01). With the exception of factor VIII-coagulant, the concentrations of hepatic synthesized proteins did not deviate from normal values in hyperthyroid and hypothyroid patients. Significant correlations were found between serum-free thyroxine on the one hand and the plasma concentrations of fibronectin (p less than 0.005), von Willebrand factor (p less than 0.001) and AVP (p less than 0.0001). Similarly, there was significant correlation between the plasma concentrations of AVP on the one hand and fibronectin (p less than 0.002) and von Willebrand factor (p less than 0.01). The results demonstrate elevated plasma levels of AVP in hyperthyroid patients and an increase during levothyroxine treatment of hypothyroid patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Plasma vasopressin responses in postpartum hypopituitarism: impaired response to osmotic stimuli.
The neurohypophyseal function was assessed in a group of 15 patients with postpartum hypopituitarism by measuring plasma arginine-vasopressin concentrations during 5% hypertonic saline infusion. None of the patients had symptoms of diabetes insipidus and all patients were on adequate cortisone and thyroxine replacement therapy before testing. The mean basal plasma vasopressin value in the patients (0.6 +/- 0.1 pmol/l) was significantly lower than that in the normal subjects (2.9 +/- 0.3 pmol/l; p < 0.01), whereas the mean serum sodium, plasma osmolality, plasma renin activity and serum aldosterone values were similar in the two groups. During the osmolar load (5% hypertonic saline), the patients revealed varying degrees of arginine-vasopressin responses to the increase in plasma osmolality. Three patients showed normal arginine-vasopressin responses, 10 had subnormal responses, and 2 had no response. During the dehydration test, the patients revealed significantly lower maximum urine osmolalities (p < 0.0025) with significantly higher concurrent mean plasma osmolality (p < 0.0025) than the controls. None of the patients showed overt polyuria at the time of the study. The results indicate the impaired osmoregulation of arginine-vasopressin secretion to an osmolar stimuli in patients with postpartum hypopituitarism, suggesting neurohypophyseal damage. In patients with Sheehan's syndrome, partial diabetes insipidus seems to be much more frequent than previously believed.
Adult↗
The promoter of the CD11b gene directs myeloid-specific and developmentally regulated expression.
Human CD11b/CD18 (complement receptor type 3) is a member of the beta 2 integrin subfamily which also includes the heterodimers CD11a/CD18 and CD11c/CD18. The CD11 molecules and the common CD18 are the products of different genes that exhibit distinct though overlapping patterns of tissue- and developmental-specific expression. Whereas expression of CD11b and CD11c is almost exclusively restricted to cells of the myeloid lineage, that of CD11a and CD18 is panleukocytic. To begin to understand the mechanisms by which expression of these gene products is restricted to leukocytes and leukocyte subpopulations and to elucidate the mechanisms by which their expression is coordinated, we have cloned and characterized the promoter region of the CD11b gene. A single transcription initiation site has been identified and the region extending 242 base pairs upstream and 71 base pairs downstream of this site has been shown to be sufficient to direct tissue-, cell-, and development-specific expression in vitro, which mimics that of the CD11b gene in vivo. Within this region there are potential binding sites for transcription factors known to be involved in hematopoietic-specific and phorbol ester-inducible gene expression. Further analysis of this region of the CD11b gene and comparison with the promoters of the CD11a, CD11c, and CD18 genes should lead to significant insights into the molecular mechanisms by which these genes are regulated during hematopoietic development and upon activation.
Antigens, CD↗
Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation.
We have isolated 5' cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.
Amino Acid Sequence↗
Expression of a soluble and functional form of the human beta 2 integrin CD11b/CD18.
Polymorphonuclear cells and monocytes (phagocytes) are a critical component of host defense against infections. However, these cells also play a significant role in host tissue damage in many noninfectious diseases, such as ischemia-reperfusion injury syndromes and rejection of transplanted organs. The leukocyte adhesion molecule family CD11/CD18 (beta 2 integrins) is critical to the function of polymorphonuclear cells and monocytes in inflammation and injury. Inherited deficiency of CD11/CD18 impairs phagocyte chemotaxis, adhesion and transmigration across endothelium, and clearance of invading microorganisms through phagocytosis and cell-mediated killing. Furthermore, murine monoclonal antibodies directed against the CD11b/CD18 (CR3) heterodimer have been shown to reduce, by 50%-80%, phagocyte-mediated ischemia-reperfusion injury in several organ systems, such as the myocardium, liver, and gastrointestinal tract and to inhibit development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice. Expression of CD11b/CD18 in a soluble and functional form might therefore be potentially useful as an anti-inflammatory agent. We have now expressed a recombinant soluble heterodimeric form of this human beta 2 integrin, normally expressed as two noncovalently associated membrane-bound subunits. The secreted receptor exhibited direct and specific binding to its ligand, iC3b, the major complement C3 opsonin, and inhibited binding of polymorphonuclear cells to recombinant interleukin 1-activated endothelium.
Amino Acid Sequence↗
Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction.
Neutrophils (PMN) migrate across intestinal epithelia in many disease states. Although such migration serves as a histological index of disease activity, little is known concerning the molecular events underlying PMN-intestinal epithelial interactions. We have studied chemotactic peptide-driven movement of PMN across cultured monolayers of the human intestinal epithelial cell line T84. Using a transmigration microassay, we show that both the decreased transepithelial resistance (76 +/- 3%) and transmigration (4 +/- 0.6 x 10(5) PMN.cm-2, when PMN applied at 6 x 10(6).cm-2) are largely prevented by MAbs which recognize either subunit of the PMN surface heterodimeric adhesion glycoprotein, CD11b/CD18. In contrast, such PMN-epithelial interactions are unaffected by MAbs recognizing either of the remaining two alpha subunits CD11a or CD11c. PMN from a leukocyte adherence deficiency patient also failed to migrate across epithelial monolayers thus confirming a requirement for CD11/18 integrins. By modifying our microassay, we were able to assess PMN transmigration across T84 monolayers in the physiological direction (which, for technical reasons, has not been studied in epithelia): transmigration was again largely attenuated by MAb to CD18 or CD11b (86 +/- 2% and 73 +/- 3% inhibition, respectively) but was unaffected by MAb to CD11a, CD11c. For standard conditions of PMN density, PMN transmigration in the physiological direction was 5-20 times more efficient than in the routinely studied opposite direction.
Antibodies, Monoclonal↗
Vasoactive intestinal polypeptide and thyrotropin-releasing hormone stimulate newly synthesized, not stored, prolactin.
Experiments were designed to determine whether vasoactive intestinal polypeptide (VIP), reported to stimulate basal PRL secretion, affects PRL processing by lactotrophs. Initially, rat anterior pituitary quarters were incubated for 2 h with [3H]leucine, with and without 10(-5) M VIP, and immunoreactive and immunoprecipitable rPRL were measured during 56 mM KCl perifusion to determine total and 3H-labeled PRL, respectively. Inclusion of VIP increased immunoreactive PRL (P less than 0.05), decreased immunoprecipitable PRL (P less than 0.01), and, therefore, decreased the specific activity of labeled PRL (P less than 0.001). These results suggested an enhanced release of newly synthesized PRL before KCl depolarization, thus decreasing the release of labeled PRL. To discriminate between the two PRL pools, newly synthesized and storage, pituitary quarters were incubated with and without 10(-5) M VIP for 4 h with [14C]leucine, 2 h in cold medium and 2 h with [3H]leucine. Immunoprecipitable PRL was measured during perifusion with 56 mM KCl. Data were depicted as the 3H/14C disintegrations per min ratio of PRL released/3H/14C disintegrations per min of total tissue to account for any differences in tissue labeling. This ratio was greater for tissue labeled in the presence of VIP (P less than 0.002). To determine whether VIP, as a secretagogue, differentiates between the newly synthesized and storage pools, VIP was added after pulse chase, as previously described. No preferential release was observed between the two groups. Finally, using the same [3H]- and [14C]leucine-labeling protocol with and without 10(-5) M VIP, tissue was perifused with medium 199 for 1 h, with 10(-5) M TRH for 30 min, with medium 199 for 30 min, and with 56 mM KCl for 1 h. Inclusion of VIP increased the 3H/14C released/3H/14C total tissue ratio during basal perifusion (P less than 0.04) and TRH exposure (P less than 0.05). Within the control group, the TRH ratio was greater than basal (P less than 0.003). These experiments suggest that newly synthesized PRL is preferentially secreted over stored PRL from tissue incubated with VIP during pulse-chase labeling; however, addition of VIP as a secretagogue did not affect either PRL pool preferentially.
Animals↗
Prolactin responses to vasoactive intestinal polypeptide and thyrotropin releasing hormone in chronic renal failure.
Prolactin response to iv bolus injection of 1 micrograms/kg vasoactive intestinal polypeptide was determined in 8 patients with chronic renal failure undergoing chronic hemodialysis and in 8 normal controls, age- and sex-matched. Plasma prolactin in the patients showed a blunted response following vasoactive intestinal polypeptide injection, whereas the controls showed significantly higher mean peak prolactin value over the baseline value (p less than 0.002). The net rise (peak levels minus basal levels) in plasma prolactin and area under the curve after vasoactive active intestinal polypeptide injection in the controls were significantly greater than those in the patients (p less than 0.001). On a separate day, each individual underwent a TRH (500 micrograms) challenge with the prolactin response determined. The patients had significantly higher peak prolactin values over baseline levels (p less than 0.02) which, however, were not significantly different from those in the control group. In the patients, the peak net prolactin increments and area under the curve were significantly higher following TRH than following vasoactive intestinal polypeptide (p less than 0.05). The net prolactin increments to TRH challenge were significantly higher in the control group than in the patients (p less than 0.001). The results demonstrate the blunted prolactin response to the stimulatory effect of vasoactive intestinal polypeptide and TRH in chronic renal failure. In chronic renal failure prolactin release after vasoactive intestinal polypeptide is more blunted than after TRH. These data suggest that the responsiveness of plasma prolactin to vasoactive intestinal polypeptide is defective in these patients, though the mechanism(s) are yet to be defined.
Adult↗
Antigen-specific radioimmunoassays for anti-neutrophil cytoplasmic antibodies in the diagnosis of rapidly progressive glomerulonephritis.
Circulating anti-neutrophil cytoplasmic antibodies (ANCA) have been described in most patients with "pauci-immune" necrotizing and crescentic glomerulonephritis. A 29-kDa serine protease (p29 or proteinase 3) and myeloperoxidase are the two best characterized antigens recognized by ANCA. The study presented here was conducted to define the diagnostic value of assays for antibodies against these two antigens in rapidly progressive glomerulonephritis. Radioimmunoassays were developed for anti-p29 and anti-myeloperoxidase antibodies, with purified antigens, and the results of the radioimmunoassays were compared with those obtained by immunofluorescence tests for ANCA. We performed assays on serum samples from 123 patients with the syndrome of rapidly progressive glomerulonephritis, as well as from 200 blood bank donors and from 717 additional control patients. Without knowledge of the results of ANCA tests, the renal pathologic findings in the 123 patients with rapidly progressive glomerulonephritis were analyzed, and 42 were classified as pauci-immune necrotizing and crescentic glomerulonephritis, 18 were classified as anti-glomerular basement membrane nephritis and 63 were classified as other forms of renal disease. We found radioimmunoassays to be more reliable in the diagnosis of pauci-immune necrotizing and crescentic glomerulonephritis than immunofluorescence testing. By radioimmunoassay, ANCA were found in 40 of 42 patients (95% sensitivity) with pauci-immune necrotizing and crescentic glomerulonephritis (14 with anti-p29 and 26 with anti-myeloperoxidase antibodies). The tests for antibodies to p29 and myeloperoxidase were 99.9 and 99.5% specific for pauci-immune necrotizing and crescentic glomerulonephritis, respectively. In the setting of rapidly progressive glomerulonephritis, a positive radioimmunoassay for anti-p29 or anti-myeloperoxidase antibodies (together with a negative test for anti-GBM antibodies) gives a probability of pauci-immune necrotizing and crescentic glomerulonephritis of over 99%.
Autoantibodies↗
Identity of Wegener's autoantigen (p29) with proteinase 3 and myeloblastin.
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Amino Acid Sequence↗
Structure and function of the leukocyte adhesion molecules CD11/CD18.
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Antigens, CD↗