with ulcerative colitis, three patients with Crohn's disease and seven control subjects, using radio- iodinated C3 prepared from fresh human plasma. Both the ...
Clin. exp. Immunol. (1977) 28, 490-495.
C3 metabolism in ulcerative colitis and Croln's disease H. J. F. HODGSON,* B. J. POTTER & D. P. JEWELL Academic Department of Medicine, Royal Free Hospital, London NW3 (Received 22 November 1976) SUMMARY
The metabolism of the third component of complement (C3) has been investigated in four patients with ulcerative colitis, three patients with Crohn's disease and seven control subjects, using radioiodinated C3 prepared from fresh human plasma. Both the fractional catabolic rate and synthesis rate of C3 were increased in the patients with inflammatory bowel disease, although the serumC3 levels were normal or raised. The results suggest that complement activation may play a role in the pathogenesis of mucosal inflammation in these diseases.
INTRODUCTION Immune mechanisms may be important in the pathogenesis of ulcerative colitis and Crohn's disease. Antibodies against gut-associated antigens are found in the diseased mucosa and in the serum of these patients (Monteiro et al., 1971; Lagercrantz et al., 1966). Immune complexes have been reported in the sera of patients with inflammatory bowel disease by a number of techniques (Jewell & MacLennan, 1973; Doe, Brown & Booth, 1973; Hodgson, Potter & Jewell, 1976), and may be present within the diseased mucosa (Ballard & Shiner, 1974). Their significance in the pathogenesis of mucosal damage is unknown, but it is possible that they initiate or maintain inflammation by activating complement. In patients with ulcerative colitis and Crohn's disease, serum-complement levels rise when disease is active; the pattern of depressed levels of C3 and other components of the classical pathway typical of immune-complex diseases, such as systemic lupus erythematosus, is not found (Thayer & Spiro, 1963; Ward & Eastwood, 1974). However, the serum concentration of a protein is the resultant of a number of influences, which include plasma volume, extravascular distribution, fractional catabolic rate and synthesis. Hypercatabolism of C3 in the presence of normal serum concentrations has been demonstrated in various diseases (Ruddy et al., 1975). The present study investigated the metabolism of C3 in patients with ulcerative colitis and Crohn's disease. PATIENTS AND METHODS Patients. Four patients with ulcerative colitis and three with Crohn's disease were studied. Details of these patients are given in Table 1. The severity of their disease was assessed using, for the patients with ulcerative colitis, the criteria of Truelove & Witts (1955), and for those with Crohn's disease, the criteria of de Dombal et al. (1974). Five patients were on no treatment; in the other two patients, treatment was not altered immediately before or during the investigation. Seven control subjects (five males, two females) comprising four healthy subjects and three hospitalized patients with non-inflammatory disorders (ischaemic heart disease and irritable bowel syndrome) were also studied. Their ages ranged from 19-58 years. All subjects studied gave informed consent to the procedure. Isolation of C3. Metabolically functional C3 was isolated from fresh human plasma by a modification of the method of Nilsson & Muller-Eberhard (1965). After precipitation of the euglobulin fraction by dialysis against 0-008 M EDTA buffer pH 5 5, and flotation of the lipoproteins from this fraction by ultracentrifugation, the material was subjected to ion-exchange chromatography on Whatman DE52 column equilibrated with 0 03 M sodium-phosphate buffer pH8-1, and washed with 700-900 ml of this buffer. Gradient elution was then started from a cone-sphere gradient-former consisting of IL 0*03 M sodium-phosphate buffer (sphere) and 500 ml 0 33 M sodium dihydrogen phosphate. The fractions found to contain C3 * Present address: Division of Gastroenterology, Massachusetts General Hospital, Boston, U.S.A. Correspondence: Dr D. P. Jewell, Academic Department of Medicine, Royal Free Hospital, London NW3.
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TABLE 1. Details of patients with inflammatory bowel disease studied
GB JD AB BB BW PM JB
Sex
Age
Diagnosis
M M M M F M M
42 38 22 48 27 18 23
Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Crohn's disease Crohn's disease Crohn's disease
Extent/site Distal Total Distal Distal Ileal Ileal Ileal
Severity Moderate Moderate Mild Moderate Moderate Moderate Moderate
Treatment None Corticosteroids None Salazopyrine None None None
activity, as judged by immunodiffusion against monospecific antiserum (Hyland, Travenol Laboratories) were then pooled and loaded, without preceding dialysis, onto a 30 cm x 4 cm2 hydroxylapatite column equilibrated with 8 mmho sodiumpotassium-phosphate buffer, pH 7 9. (Hydroxylapatite as prepared by the method of Tiselius et al., 1956). After washing with 500 ml of starting buffer, C3 was then eluted using a linear conductivity gradient, with the same buffer, between 8 and 20 mmho. C3 activity was eluted between 14 and 17 mmho. These fractions were then quickly concentrated by ultrafiltration. All buffers were prepared in water for injection, and contained either streptomycin sulphate (100 mg/l) and benzyl penicillin (l x 106 u/I) or gentamycin sulphate (80 mg/l) and the sodium salt of ampicillin (250 mg/l). All apparatus and buffers were either autoclaved or sterilized using commercial sterilising agents. Functional activity of the C3 preparations was determined using the C3-complement-test system from Cordis Laboratories (Miami, Florida). Results were expressed as percentage functional activity of pooled control sera. Radioiodine labelling of C3. C3 was labelled with 12
