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In Systemic Lupus Erythematosus Ann Rheum Dis: first published as 10.1136/ard.39.4.312 on 1 August 1980. Downloaded from Annals of the Rheumatic Diseases, 1980, 39, 312-317 Evidence of an immediate hypersensitivity mechanism in systemic lupus erythematosus J. EGIDO, M. SANCHEZ CRESPO, C. LAHOZ, R. GARCIA, M. LOPEZ-TRASCASA, AND L. HERNANDO From the Renal and Immunology Laboratories, Fundacio'n Jimnunez Diaz, Madrid SummARY In 30 patients with systemic lupus erythematosus the number of circulating basophils was counted in different stages of activity. An inverse correlation was found between the absolute basophil count and anti-DNA antibodies and presumptive circulating immune complexes (as judged by polyethylene glycol precipitation of serum). A positive correlation was found between the absolute basophil count and C3 or C4 levels. IgE on the basophil surface was determined by radioimmunoassay in 7 patients. All of them showed a significantly high surface IgE number. When the count of circulating basophils was roughly normal, 5 out of the 6 patients showed a positive basophil degranulation test with native DNA. These results suggest the existence of an anti-DNA specific IgE in lupus patients. Depression of the circulating basophil count may be a useful index of lupus activity. copyright. In the acute serum sickness of rabbits the deposition Materials and methods of immune complexes in the blood-vessel walls involves an increase in vascular permeability induced We studied 30 patients with systemic lupus ery- by the release ofvasoactive amines from the platelets.' thematosus, who fulfilled diagnostic clinical criteria http://ard.bmj.com/ In these animals the occurrence of the glomerular of the American Rheumatism Association (ARA) lesions is particularly associated with antigen- and certain immunological criteria (antinuclear dependent histamine release from leucocytes2; and, antibodies, anti-DNA antibodies and low serum on addition of the antigen, basophils sensitised complement). All patients had renal involvement, with IgE degranulate and release also a platelet- shown by renal biopsy, confirmed by light micro- activating factor (PAF) which aggregates platelets scopy, immunofluorescence, and in some cases and releases their histamine.3 We have recently electron microscopy. shown that a diminution of circulating basophils on September 26, 2021 by guest. Protected precedes the onset of proteinuria in acute experi- STAINING AND COUNTING OF BASOPHILS mental serum sickness; and those rabbits whose The technique has been described previously in basophil count remained normal showed no pro- rabbits,9 but it has also been found useful for human teinuria, although they had circulating immune basophil counting.'0 Briefly, blood and dye were complexes bigger than 19 S in size.4 mixed in a 1/10 proportion and placed in both The role of immediate hypersensitivity in immune chambers of a Fuchs-Rosenthal cell. Cells were complex diseases in man has received little atten- counted at a magnification of 200. With this tech- tion.5-8 Accordingly, the purpose ofthis work was to nique basophils were the only cells with stained study the implication of the IgE-basophil system in cytoplasm. The counts were performed by 2 persons, systemic lupus erythematosus, a prototype of neither of them knowing the clinical situation or the immune complex disease in man. treatment received by the patients. Samples were drawn in the morning. Average absolute basophil counts and percentage of basophils in relation to the Accepted for publication 22 August 1979. total leucocyte count in 47 control subjects were Correspondence to Dr J. Egido, Laboratorio de Nefrologia, ± ± 0 34 Fundaci6n Jim6nez Dfaz, Avda. Reyes Cat6licos 2, Madrid- respectively 44 5 15 (SD)/mm3 and 0*51 3, Spain. (SD). 312 Ann Rheum Dis: first published as 10.1136/ard.39.4.312 on 1 August 1980. Downloaded from Evidence ofan immediate hypersensitivity mechanism in systemic lupus erythematosus 313 BASOPHIL DEGRANULATION UTILISING POLYETHYLENE GLYCOL (PEG) PRECIPITATION SONICATED DNA FOR DETECTION OF PRESUMPTIVE This was undertaken in 6 patients during periods IMMUNE COMPLEXES when their basophil count were within normal limits. Diluted sera were mixed in equal portions with Since native DNA preparations are viscous, due to PEG (6000 Merck) solution (borate buffer) at a their high molecular weight, these are technically final PEG concentration of 355%.16 The washed unsuitable for degranulation studies. Accordingly, precipitate was dissolved in sodium hydroxide, and calf thymus DNA (Sigma type 1) at a concentration the quantity of precipitated protein present was of 10 ,ug/ml was sonicated in an ice bath using a measured by optical density (OD) at 280 nm. The Branson W185 sonicator for 40 seconds at maximum threshold ofpositivity was taken as 0 * 130 OD, which output in order to obtain uniform fragments of represents two standard deviations above the mean approximately 106 daltons.11 This DNA was con- value for 60 normal controls. The higher values were sidered immunologically as native DNA (n-DNA) considered positive, the lower values negative. because in inhibition studies (kindly performed by Dr Picazo) it was totally unreactive with rabbit COMPLEMENT antisera to denatured DNA.12 Whole blood of lupus Serum levels of C3, C4, and C3PA were measured by patients (10 ml in tubes with heparin) was distributed radial immunodiffusion, following Mancini's tech- in 5 ml plastic tubes in 1 ml aliquots. 10 Vul of a DNA nique17 (Behring Partigen plates). solution in Tris buffer, 0' 2M, pH 8, at final con- centrations of 0 01, 0.1, 1 and 10 Vg, were added. Results Two tubes were used as controls. One contained 10 Vg of unrelated protein (bovine serum albumin) and the other contained neither antigen nor BSA. CIRCULATING BASOPHIL COUNT AND ITS RELATIONSHIP WITH ANTI-DNA Tubes were placed at 370C under constant motion copyright. for 15 minutes, after which 25 ,ul of 0-2 M EDTA, ANTIBODIES, SERUM C3, C4, AND C3PA pH 7 4, was added to stop the reaction, and the LEVELS, AND PEG PRECIPITATION samples were placed in a rotator. Each sample was Seventy-one determinations were made in the 30 counted twice and the percentage of basophil lupus patients in different stages of disease activity. degranulation was calculated from the following The average absolute basophil count and the formula: percentage of basophils in relation to the total leucocytes in lupus patients was of 16 08 17 Mean no. of basophils in control samples -mean no. ± basophils in test x 100. (SD)/mm3 and 0 30 ± 0 23, significantly low with http://ard.bmj.com/ Mean no. of basophils in control samples respect to controls (P<0.0005 and P<0 005 To show that degranulation is calcium-dependent respectively). The basophil count in 67 nonlupus and therefore meaningful, the degranulation test patients with biopsy evidence of glomerulonephritis was repeated at optimum DNA concentration in (minimal changes, mesangiocapillary, membranous, the presence of 5 mM EDTA. and IgA mesangial glomerulonephritis) has not shown significant differences from the controls (data MEASURE OF IgE ON THE BASOPHIL SURFACE not shown). on September 26, 2021 by guest. Protected IgE on the basophil surface was determined on 7 In order to study whether a depressed circulating lupus patients at different times by a radioimmuno- basophil count represented another sign of lupus assay method previously described.13 The assay activity this parameter was compared with some system was based on the capacity of unlabelled immunological data generally considered to be immunoglobulin to inhibit the reaction between 'markers' of activity. Correlations emerged between labelled IgE protein and anti-IgE.14 circulating basophil count and DNA antibodies (expressed as DNA-binding percentage) and serum ANTI-DNA ANTIBODY DETERMINATION C3 and C4 levels; these relationships are shown in The method used was that of precipitation with 50% Figs. 1, 2, and 3, respectively. No correlation was saturated ammonium sulphate, using Escherichia found with serum C3PA levels. The relationship Coli DNA labelled with 14C, following Farr's between basophil counts and PEG precipitation technique.15 Binding values under 30% were con- in 28 patients is shown in Fig. 4. In those patients sidered normal. Sera were heated at 560C for 30 having a positive precipitation with PEG the average minutes to eliminate nonspecific binding of DNA basophil count was 9-42 ± 6-95 (SD)/mm3, while to Clq. All readings were made in duplicate, and a it was 19 40 ± 14.87/mm3 in those having it control serum with a known binding value was negative (P<0-001). A significant correlation was included in each test. also found when we compared the average values Ann Rheum Dis: first published as 10.1136/ard.39.4.312 on 1 August 1980. Downloaded from 314 Egido, Sdnchez Crespo, Lahoz, Garcia, Lopez-Trascasa, Hernando E SD of the percentage of basophils in relation greater than 30% (Fig. 5). Maximal degranulation to the total leucocytes (0-20 ± 0-14 versus 0 44 + was reached at 1 Vg DNA/ml, except in the patient.' 0 31) in these 2 groups. In the majority of the patients the dose-response curves were similar to those obtained by the same CIRCULATING BASOPHIL SENSITISATION technique in patients sensitised against different WITH RESPECT TO NATIVE DNA drugs.10 In all patients basophil degranulation was Five out of 6 patients whose basophils were incu- inhibited when the test was performed in the pre- bated with sonicated DNA showed degranulation sence of 5 mM EDTA. % 6DNA bindinq r =-0,552 p <o,oo1 Fig. 1 Correlation between the number ofcirculating basophils stained by a modified toluidine blue and the DNA antibodies, expressed as DNA-binding percentage. copyright. 5 10 15 45 So Basoph/s/lMm3 http://ard.bmj.com/ C'3 mg%. .470 * 200 on September 26, 2021 by guest. Protected Fig. 2 Correlation between the number of circulating basophils r= 0,557 and serum C3 p< 0,00f ,40.
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