THE ALTERNATE COMPLEMENT PATHWAY IN INFLAMMATORY BOWEL DISEASE

Transcription

1 GASTROENTEROWGY 70: , 1976 Copyright 1976 by The Williams & Wilkins Co. Vol. 70, No.2 Printed in U.S.A. THE ALTERNATE COMPLEMENT PATHWAY IN INFLAMMATORY BOWEL DISEASE Quantitation of the C3 proactivator (factor B) protein PETER A. FEINSTEIN, M.M.S., M.D., STEPHEN R. KAPLAN, M.D., AND WALTER R. THAYER, JR., M.D. Department of Medicine, Brown University, The Roger Williams General Hospital and the Rhode Island Hospital, Providence, Rhode Island A component of the complement system's alternate pathway was investigated in ulcerative colitis and Crohn's disease. The mean (Factor B) titer in normals was 74 ± 15%; in ulcerative colitis, 92 ± 18%; and in Crohn's disease, 119 ± 24%. Significance was at the P < level when the mean values for the ulcerative colitis and the Crohn's disease groups were compared to normal subjects. Titers did not change significantly with exacerbation or amelioration of the diseases or when patient groups were analyzed according to the mode of treatment received. The complement system is an important final common pathway of tissue injury in many inflammatory diseases. The "classical" pathway of complement activation is initiated by an antigen-antibody complex, which activates components C1, C4, and C2, to form an enzymatic product that cleaves C3 to its active state. As a result of the activation of C3 through C9, important biologically active products are released, such as anaphylatoxins and chemotactic factors. Ultimately the disruption of biomembranes causing cytolysis or tissue injury may occur. 1 In recent years it has been appreciated that another set of proteins comprising the "alternate" or "properdin" pathway is capable of ~ c t i v ac3,2:5 t i nthus g bypassing the initial antigen-antibody and C1, C4, and C2 reactions, but still eliciting the important biological functions of the system. 6-8 Substances known to be capable of initiating this pathway include endotoxic lipopolysaccharides, aggregated myeloma proteins IgA 1, IgA 2, IgG 4,3 aggregated IgE,12 cobra venom factor,3. 13 and yeast cell wall, zymosan. 14, 15 Investigations of patients with active ulcerative colitis (UC) have demonstrated an elevation of serum total hemolytic complement activity An increased incidence of precipitin reactions to the C1q component has been found in sera of patients with Crohn's disease (CD) and UC, 19 and there is an abnormality in localizations of the C3 component in the rectal mucosa of patients with UC.20, 21 Serum C3 and C4 component levels have been shown to be elevated in both conditions. 22 Because bacteriallipopolysaccharides have been sug- Received April ~. 19'15. Accepted July This work was supported by a grant from the National Foundation for Ileitis and Colitis and from the National Arthritis Foundation. Dr. Feinstein's work was performed as partial fulfillment for a M.M.S. Degree at Brown University. Address reprint requests to Dr. Walter Thayer, Rhode Island Hospital, Providence, Rhode Island gested to be of importance in the pathogenesis of the inflammatory bowel diseases 23 and because they also can activate the complement system's alternate pathway, a component of this properdin pathway was investigated in UC and CD. Materials and Methods Adult sera from 17 healthy volunteers were obtained from the blood bank within 20 hr of donation. The blood was allowed to clot and then centrifuged. The resulting sera were stored at 4 C during the period between centrifugation and pickup (3 to 4 hr) and were subsequently stored at -90 C until used. One normal sample was initially designated as the reference serum for the protocol. This then served as a standard to which all results were compared. Blood from 45 patients, 14 with CD, 26 with DC, and 5 with DC treated by surgery (total colectomy and ileostomy), who were either hospitalized or being seen in the Gastrointestinal Outpatient Clinic, was processed in similar fashion. The diagnosis in each case was made in accordance with previously established criteria. 24 Several patients were able to donate serial blood samples over periods of time ranging up to 5 months, to aid in the correlation of findings to changes in clinical disease status or to changes in therapeutic regime. The alternate pathway was investigated by measuring serum levels of one of its components, the C3 proactivator () protein. levels were assayed by use of the radial immunodiffusion technique of Mancini, as described by Ruddy et al. 25 The anti-human rabbit serum, lot 1548k, was obtained from Behring Diagnostic Laboratories, Somerville, New Jersey, and stored at 4 C. Plates were made of 3% Noble agar and buffer, with antiserum added in an amount resulting in a 1:50 dilution. The buffer was composed of 9 g sodium Veronal, 65 ml 'of 0.1 normal HCl, and 11 ml of 0.86 EDTA, diluted to 1 liter. Each plate contained 3 ml of the agarcbuffer-antibody mixture, and 20 wells were made, each having a diameter of 1.4 mm. Each plate was duplicated. All plates contained 3 wells respectively filled with the reference serum diluted in 0.9% saline to 1/4, liz, and full strength. Plates were read after 48 hr of humid incubation at room temperature using a measuring magnifier (Bausch and Lomb). The diameter of the diffusion ring for each experimental sample was

2 182 FEINSTEIN ET AL. Vol. 70, No.2 squared and compared with a concentration curve derived from the relationship between the serial dilutions of the reference sera and the squares of the diameters of each of their diffusion rings. Individual results are expressed as a percentage of reference serum level. Results of each patient group and of controls are expressed as the mean for the group plus or minus one standard deviation. Statistical analysis utilizing the Student's t-test was applied to the groups. Results The results from the two groups of patients and the normal adults are shown in figure 1. The mean titer in the normal group was 74 ± 15%, the range being 43 to 108%. The patients with UC had a mean titer of 92 ± 18%, with the range of 69 to 140%. The CD group had a mean serum titer of 119 ± 24%, the range being 77 to 183%. For the inflammatory bowel diseases as a whole, the mean was 100 ± 21 %. These differences in the means were all highly significant: for the inflammatory bowel diseases as a whole compared to the normal group, P < 0.001; for the UC patients compared with normals, P < 0.001; for the CD patients compared with normals, P < 0.001; and for the CD group compared with the UC group, P < Figure 2 demonstrates the division of the patients with CD into two clinical groups, those experiencing an exacerbation of their disease, and those who were considered to be doing well or stable at the time the sera were obtained. Because 6 CD patients contributed 2 or more samples over the course of the study, additional data points are given. The mean titer for the patients doing well was 108 ± 17%. When this group was compared with normals, the degree of significance was at the P < level. The patients experiencing an exacerbation had a mean titer of 137 ± 25%, with the difference between this value and that for normals being highly significant (P < 0.001). When the mean for those doing well or stable was compared with those (Of. ref.) 180 t I Normal mean ± I standard deviation NORMALS CROHN'S DISEASE ULCERATIVE COLITIS FIG. 1. C3 proactivator titers in normal subjects, patients with Crohn's disease, and patients with ulcerative colitis.!e, mean ± 1 SD. ('Yo ref.) O D 0 f ~ ~ Normal meari ±. I I deviation CROHN'S DISEASE WITH EXACERBATION CROHN'S DISEASE DOING WELL OR STABLE FIG. 2. The C3 proactivator titers in Crohn's disease, in patients who are in exacerbation, or who are stable or doing well.!e, mean ± 1 sp. (Of. ref.) I I 1 ~ ~! ~ ~." ~ ~ o r lmean a l ± I jtandard djvialion UNTREATED MEDICAL SALICYL- STEROIDS TREATMENT AZOSULFA- PYRIDINE FIG. 3. C3 proactivator titers in patients with Crohn's disease according to treatment. ~, mean ± 1 SD. having exacerbations, the difference was less significant (P < 0.01). In the 3 patients experiencing both exacerbation and remission while followed serially, titers were variable but conformed to the above findings. The group was too small for any meaningful relationship to be established between the variation in titer and the clinical impression of disease activity. Figure 3 presents the data from the CD group in terms of therapeutic modalities. The mean titer for those patients receiving no therapy was 129 ± 27%, whereas for those receiving therapy it was 107 ± 17 %. The differences when both of these groups were compared with the normal group were highly significant (P < 0.001); however, when values for untreated patients were compared with values for treated patients, the difference was not so significant (P = 0,05). The 3 patients receiving salicylazosulfapyridine as their therapy had a mean titer of 110 ± 9%, which was not significantly

3 February 1976 FACTOR B PROTEIN 183 different from the group of patients receiving no treatment (P < 0.1). There were not enough samples collected to determine if any change was apparent in the titer before and after salicylazosulfapyridine. Six patients with CD received treatment with steroids and exhibited a mean titer of 102 ± 20%; this, like the value for salicylazosulfapyridine-treated patients, is not significantly different from patients receiving no therapy (P < 0.1). Similar data for the patients with DC are shown in figures 4 and 5. Because 8 patients contributed more than 1 sample, additional data points are given. The mean titer for the group considered clinically as doing well or stable is 89 ± 16%, which is different from the normal (P < 0.01). The mean for the group having increased disease activity is 100 ± 20%; this value is significantly different from the normal (P < 0.001). The difference between the group doing well or stable and those with exacerbation is not significant (P > 0.1). Four patients, who experienced both exacerbation and remission while being followed serially, had variable titers. The group was too small for any relationship to be established between the variation in titers and disease activity. Figure 5 demonstrates the effect of therapy on C3P A titers in the DC group. The mean level for those receiving no treatment was 98 ± 21 %, whereas for those being treated it was 87 ± 12%. The difference between the two groups was not significant (P < 0.1). However, the values for those receiving no therapy were significantly different from the normal values (P < 0.001), whereas values for those receiving treatment were less so (P < 0.01). For the 8 patients whose treatment consisted of salicylazosulfapyridine, the mean titer was 83 ± 10%. This value was not significantly different from that for the untreated group (P < 0.02), from that for the normal group (P < 0.1), or from that for the group having an exacerbation (P < 0.05). Although this is not shown in figure 5, 4 of these patients were evaluated (0;. ref.) 3 0 ~ -L ~ ULCERATIVE COLITIS ULCERATIVE COLITIS STABLE OR WITH EXACERBATION DOING WELL FIG. 4. C3 proactivator titers in ulcerative colitis patients who are in exacerbation, or who are stable or doing well. 33 ' mean ± 1 SD. (0;. ref.) ~ ~ ~ UNTREATED MEDICAL SALICYL- STEROIDS TREATMENT AZOSULFA COLECTOMY PYRIDINE.. This patient received both salicylazosulfapyridine and steroid therapy FIG. 5. C3 proactivator titers in patients with ulcerative colitis according to treatment. 33 ' mean ± 1 SD. before and after commencing salicylazosulfapyridine therapy. The pretreatment mean titer was 95 ± 15%; that for post-treatment, 86 ± 9%. A significant difference could not be determined (P > 0.1). The mean for the 5 patients who were given steroids was 96 ± 10%. Compared with the value for untreated patients, this was not significantly different (P > 0.1), nor was it significantly different from the exacerbation group (P > 0.1). When compared with the normal group, however, a difference was observed (P < 0.01). Five patients were treated surgically as a result of their disease (total colectomy and ileostomy). As a group, patients having undergone surgical intervention manifested a mean titer of 93 ± 12%. This value was not significantly different from either the untreated group (P > 0.1), or the exacerbation group (P > 0.1), although some significance was determined (P < 0.01) when it was compared with the value for normal subjects. Discussion The protein is one of several components of an alternate pathway of complement activation. The complete reaction sequence of this pathway and the importance of each of its components remain a subject of investigation. is one of the final proteins in the alternative mechanisms; its activation results in the generation of split products with {3 and 'Y mobilities. The fragment with 'Y mobility, called the C3 activator, contains C3 convertase activity3 and causes depletion of the late acting complement components without activation of C1, C4, and C2. The results presented in figures 1 through 5 demonstrate that there is a disturbance in the metabolism of this component in both CD and DC, but that this abnormality is slightly different in each. Significantly elevated levels of serum were present in both groups of patients with CD, those in remission, and those doing poorly. In the DC group, those patients with

4 184 FEINSTEIN ET AL. Vol. 70. No.2 exacerbation of disease had significantly elevated levels, whereas those in apparent remission differed less m,arkedly from the control group. These findings agree with previously published results. Thayer and Spiro 16 investigated serum complement levels in 15 patients with DC. Total hemolytic complement titer was within the normal range for 10 of 15 patients who were in a remissive phase of the disease. The remaining 5 patients had levels clearly above normal, and of these, 4 were designated as acutely ill; the other was asymptomatic. A similar study by Fletcher17 found hemolytic complement levels to be normal or high in 14 of 15 patients who were systemically ill with DC. Fogel et al. 18 investigated serum hemolytic complement titers in patients with DC compared with a range for normal controls of 154 to 250 C'R50 per ml and a mean of 200. It was found that the mean C'R50 per mllevel of 16 hospitalized patients with active DC was 270, ranging from 180 to 254 C'R50 per ml. Only 2 patients had C'R50 levels lower than the mean for normal controls. Ward and Eastwood 22 studied the individual complement components C3 and C4. Significantly elevated C4 levels were found in CD patients, including those in apparent remission. In the DC group, C4 levels were significantly raised only in those cases in exacerbation; those in remission did not differ from controls. A similar although less exaggerated disturbance was noted in C3 levels. The conclusions from these findings are 3-fold: 1) there is a general derangement in complement metabolism in DC reflected in the abnormal C'R50 (there are no data on CD); 2) this general derangement is paralleled by a similar pattern of abnormalities in the component parts of both the classical (C3, C4) and alternate () pathways in CD and DC; and 3) there is a difference in complement metabolism in both classical and alternate pathway components between DC and CD patients in remission. An explanation for these conclusions would require a kinetic study of the individual components. This study and that by Ward 22 do not indicate whether component levels are elevated because of lack of consumption (i.e., classical and alternate pathways are turned off or have a blocked reaction sequence), or whether an inflammatory condition has caused the high titers (acute phase phenomena) despite an increased consumption rate. Such metabolic studies are required of both the classical and alternate pathways individually, before the contribution of each can be assessed. 26 Current investigation lends significance to our results in terms of possible pathogenetic roles for the alternate pathway in both DC and CD. Several studies have indicated that there are circulating immune complexes in the sera of patients with inflammatory bowel disease. 27, 28 Providing further evidence for such immune complexes,19 precipitin reactions using the Clq complement component were positive in a high proportion of patients with these diseases. The elevated titers demonstrated in this study do not support a role for the alternate pathway as a link between circulating immune complexes and the colonic cell damage characteristic of DC and CD. As mentioned, however, kinetic studies are necessary in order to render verification that C3 and are not consumed. Although limited, the results regarding serum concentration and the treatment of DC and CD also support the conclusion that the alternate pathway is not an important pathogenetic factor. In both diseases there was no significant difference between titers in treated or untreated groups. This lack of significance was maintained for individual treatment modalities: steroids, salicylazosulfapyridine, and surgery. These measures are, nevertheless, of proven benefit; thus, if the complement pathway were involved, one might expect the results to be affected by these regimens. Even the only truly controlled group, that of 4 DC patients treated with salicylazosulfapyridine, demonstrated no significant difference in titers before and after treatment. Interestingly, complement abnormalities seem to persist after total colectomy and ileostomy for C3P A titers of 5 DC patients treated by this modality as compared with normals (P < 0.01). This finding agrees at the same level of significance with Ward's22 finding for classical pathway components C3 and C4 in 13 DC patients treated with surgery. At the present time, the explanation for this phenomenon remains unclear. REFERENCES 1. Ruddy S, Gigli I, Austen KF: The complement system of man. N Engl J Med 287: , , , , Pillemer L, Blum L, Lepow IH, et al: Properdin system and immunity; demonstration and isolation of new serum protein, properdin, and its role III lmmune phenomena. Science : , G6tze 0, Muller-Eberhard HJ: The C3 activator system: An alternate pathway of complement activation. J Exp Med 134:90s-108s, Muller-Eberhard HJ, G6tze 0: C3 proactivator convertase and its mode of action. J Exp Med 135: , Lachmann PJ, Nicol P: Reaction mechanism of the alternate pathway of complement fixation. Lancet 1: , Mergenhagen SE, Snyderman R, Gewurz H, et al: Significance of complement to the mechanism of action of endotoxin. 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