Int. Archs Allergy appl. Iinniun. 54: 434-442 (1977)
Studies of C l Subcomponents in Chronic Urticaria and Angioedema Anna-Brita Laurell, Vila Mariensson and Anders G. Sjoholm Institute of Medical Microbiology, Lund
Introduction
The pathogenesis of chronic urticaria and angioedema is not well understood. Classification into different types is mainly based on clinical features and, when possi ble, on triggering factors [33], Urticaria may occur in systemic diseases, notably SLE [6] and mixed cryoglobulinemia [5], In these conditions hypocomplementemia is of ten encountered [25, 27]. In hereditary an gioedema, complement activation is thought to be the main mediator of the symptoms
[10]. Although hypocomplementemia ap pears to be rare in chronic urticaria and an gioedema, several cases have been described in which abnormally low complement com ponent levels suggested that complement ac tivation was involved in the pathogenesis [2, 19, 20, 28]. Laurell et al. [14] reported ab errations of Cl subcomponents, C4 and C3 in some patients with chronic urticaria and angioedema. Recently, complexes of Cl subcompo nents in human sera were studied with crossed immunoelectrophoresis [16], Thus,
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 6:31:50 AM
Abstract. Clq, C lr, Cls, C3, C4 and Cl IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. Abnormal Clq and Cls levels were found in about 30%> of the patients. In seven sera C lr was not measurable due to the appearance of diffuse precipitates. The levels of C3 and/or C4 were decreased in five sera with aberrations of Cl subcomponents in the electroimmunoassay. None of the patients showed reduced Cl 1A levels in the electroimmunoassay. The presence in sera of abnormal C1 subcomponent complexes was studied by crossed immunoelectrophoresis. Sera from 11% of the patients contained C lr-C ls complexes. Increased amounts of a.2 complexes (Clr-CIs-CI IA) were found in 33°/« of the patients. A major part of the Clq in sera yielding abnormal C lr precipitates had the same electrophoretic mobility as isolated Clq and was not associated with the Clqrs complex. Cl activity in hemolytic tests was low in these sera as well as in sera with decreased Clq levels. In the esterolytic assay for Cl IA low values were found in 14 patients. Repeated sampling and family studies in appropriate cases gave no evidence for genetically determined deficiencies of Clq, Cl r or Cl IA.
435
Laurell/Mártensson/Sjóholm
Materials and Methods Pathological sera. Serum samples from 150 pa tients with a clinical diagnosis of chronic urticaria or angioedema were stored in aliquots at -80 °C until analysed. In some cases repeated sampling and family studies were carried out. The sera were sent frozen to the laboratory. In a few cases chilled sera arriving within 1 day of sampling were also included. The records of the patients were reviewed. Immunochemical determination of comple ment components. The electroimmunoassay [17] was used for quantitation of Clq, C lr, Cls, C3, C4, and Cl 1A according to procedures pre viously described [29, 30]. Complement compo nent levels were given in percentage of the con centrations in a normal reference pool. Esterolytic assay for Cl IA. Determinations were made with the esterolytic assay of Levy and Lepow [18] as modified by LaurelI et at. [13]. The normal range is 18-28 U/ml. Crossed immunoelectrophoresis [8] of C lr and Cls in serum was carried out as described by Lau relI et al. [16). 0.075 M barbital buffer, pH 8.6, containing 0.002 M calcium was used during the initial separation step. The second electrophoretic step was performed with 0.075 M barbital buffer containing 0.002 M EDTA at low voltage (3-4 V/ cm) for about 18 h.
For analysis of Clq by crossed immunoelec trophoresis 0.15 M barbital buffer pH 8.6 contain ing 0.002 M calcium was used in both electropho retic steps. The initial separation was carried out for 2-3 h at 10 V/cm and the second electrophoret ic step at 3-4 V/cm for about 18 h. Estimates of a2 Cls complexes were made by measuring the heights of a2 Cls peaks and grading them from 1+ to 3 + . a2 Cls in a normal refer ence serum was recorded as negative. The az Cls peaks in several normal sera investigated varied between negative and 1 + . Purified human complement components. Clq was prepared according to Yonemasu and Stroud [34]. Functionally purified C4 and C2 were ob tained as described earlier [16]. C2 in oxidized form was used in hemolytic titrations [22]. Ma cromolecular Cl was purified from neutral euglobulin by gel filtration on Sepharose CL-6B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) in acetate buffer pH 5.5, 0.2 M NaCl, 5 mM CaCl2 [9], Functional assays for Cl. EAC4 derived from EACI4 prepared with IgM amboceptor and functionally purified Cl and C4 were used for ti trations of Cl in serum [23]. Cl titrations were also performed using EA mainly according to Opferkuch et al. [21]. Both methods gave linear do se-response curves when the calculated number of lesions per cell (z) was plotted against relative Cl concentrations. Cl levels in hemolytic tests were given in percentage of the activity in a normal ser um used as reference.
Results
Quantitative Estimation of Complement Components Clq. Clr, Cls, C3, C4 and Cl IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. The levels of these proteins in normal sera have been reported elsewhere [29, 30], The variation of Clq and Cls levels in the patients’ sera was wider than in normal sera. About 30°/o of the patients showed ab-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 6:31:50 AM
in the presence of calcium during the initial separation step, /f„ C lr-C ls complexes and a., complexes between Clr, C is and Cl IA could be distinguished from classi cal macromolecular Clqrs remaining at the application site.
Studies of C l Subcomponents in Chronic Urticaria and Angioedema Anna-Brita Laurell, Vila Mariensson and Anders G. Sjoholm Institute of Medical Microbiology, Lund
Introduction
The pathogenesis of chronic urticaria and angioedema is not well understood. Classification into different types is mainly based on clinical features and, when possi ble, on triggering factors [33], Urticaria may occur in systemic diseases, notably SLE [6] and mixed cryoglobulinemia [5], In these conditions hypocomplementemia is of ten encountered [25, 27]. In hereditary an gioedema, complement activation is thought to be the main mediator of the symptoms
[10]. Although hypocomplementemia ap pears to be rare in chronic urticaria and an gioedema, several cases have been described in which abnormally low complement com ponent levels suggested that complement ac tivation was involved in the pathogenesis [2, 19, 20, 28]. Laurell et al. [14] reported ab errations of Cl subcomponents, C4 and C3 in some patients with chronic urticaria and angioedema. Recently, complexes of Cl subcompo nents in human sera were studied with crossed immunoelectrophoresis [16], Thus,
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 6:31:50 AM
Abstract. Clq, C lr, Cls, C3, C4 and Cl IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. Abnormal Clq and Cls levels were found in about 30%> of the patients. In seven sera C lr was not measurable due to the appearance of diffuse precipitates. The levels of C3 and/or C4 were decreased in five sera with aberrations of Cl subcomponents in the electroimmunoassay. None of the patients showed reduced Cl 1A levels in the electroimmunoassay. The presence in sera of abnormal C1 subcomponent complexes was studied by crossed immunoelectrophoresis. Sera from 11% of the patients contained C lr-C ls complexes. Increased amounts of a.2 complexes (Clr-CIs-CI IA) were found in 33°/« of the patients. A major part of the Clq in sera yielding abnormal C lr precipitates had the same electrophoretic mobility as isolated Clq and was not associated with the Clqrs complex. Cl activity in hemolytic tests was low in these sera as well as in sera with decreased Clq levels. In the esterolytic assay for Cl IA low values were found in 14 patients. Repeated sampling and family studies in appropriate cases gave no evidence for genetically determined deficiencies of Clq, Cl r or Cl IA.
435
Laurell/Mártensson/Sjóholm
Materials and Methods Pathological sera. Serum samples from 150 pa tients with a clinical diagnosis of chronic urticaria or angioedema were stored in aliquots at -80 °C until analysed. In some cases repeated sampling and family studies were carried out. The sera were sent frozen to the laboratory. In a few cases chilled sera arriving within 1 day of sampling were also included. The records of the patients were reviewed. Immunochemical determination of comple ment components. The electroimmunoassay [17] was used for quantitation of Clq, C lr, Cls, C3, C4, and Cl 1A according to procedures pre viously described [29, 30]. Complement compo nent levels were given in percentage of the con centrations in a normal reference pool. Esterolytic assay for Cl IA. Determinations were made with the esterolytic assay of Levy and Lepow [18] as modified by LaurelI et at. [13]. The normal range is 18-28 U/ml. Crossed immunoelectrophoresis [8] of C lr and Cls in serum was carried out as described by Lau relI et al. [16). 0.075 M barbital buffer, pH 8.6, containing 0.002 M calcium was used during the initial separation step. The second electrophoretic step was performed with 0.075 M barbital buffer containing 0.002 M EDTA at low voltage (3-4 V/ cm) for about 18 h.
For analysis of Clq by crossed immunoelec trophoresis 0.15 M barbital buffer pH 8.6 contain ing 0.002 M calcium was used in both electropho retic steps. The initial separation was carried out for 2-3 h at 10 V/cm and the second electrophoret ic step at 3-4 V/cm for about 18 h. Estimates of a2 Cls complexes were made by measuring the heights of a2 Cls peaks and grading them from 1+ to 3 + . a2 Cls in a normal refer ence serum was recorded as negative. The az Cls peaks in several normal sera investigated varied between negative and 1 + . Purified human complement components. Clq was prepared according to Yonemasu and Stroud [34]. Functionally purified C4 and C2 were ob tained as described earlier [16]. C2 in oxidized form was used in hemolytic titrations [22]. Ma cromolecular Cl was purified from neutral euglobulin by gel filtration on Sepharose CL-6B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) in acetate buffer pH 5.5, 0.2 M NaCl, 5 mM CaCl2 [9], Functional assays for Cl. EAC4 derived from EACI4 prepared with IgM amboceptor and functionally purified Cl and C4 were used for ti trations of Cl in serum [23]. Cl titrations were also performed using EA mainly according to Opferkuch et al. [21]. Both methods gave linear do se-response curves when the calculated number of lesions per cell (z) was plotted against relative Cl concentrations. Cl levels in hemolytic tests were given in percentage of the activity in a normal ser um used as reference.
Results
Quantitative Estimation of Complement Components Clq. Clr, Cls, C3, C4 and Cl IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. The levels of these proteins in normal sera have been reported elsewhere [29, 30], The variation of Clq and Cls levels in the patients’ sera was wider than in normal sera. About 30°/o of the patients showed ab-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 6:31:50 AM
in the presence of calcium during the initial separation step, /f„ C lr-C ls complexes and a., complexes between Clr, C is and Cl IA could be distinguished from classi cal macromolecular Clqrs remaining at the application site.
![[Histaminergic angioedema and chronic urticaria].](/assets/img/hold.png)
