Immunology 1979 37 467
Inhibition of antibody-dependent cellular cytotoxicity by artificial immune complexes and pathological sera
J. M. T. HOWAT*, M. MOORE, ANGELA M. HILTON& I. KIMBERImmunology Department, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 18 October 1978: acceptedfor publication 6 November 1978
subjects was thus expressed as a function of ADCC in a standard batch of foetal bovine serum (FBS). Under these conditions differences between pathological (n= 51) and normal (n= 52) sera were highly significant (P < 0 001), which could not be explained by the presence in the patients' sera of H L-A antibodies reactive with the effector cells, nor by a deficit in nutritional support of ADCC. The absence of a correlation between inhibition of ADCC and total serum IgG or IgM inferred that inhibition was attributable to immune complexes in the IBD sera. The limitations of this assay for assessment of the incidence of immune complexes in pathological sera are discussed.
Summary. Detection of immune complexes by inhibition of antibody-dependent cellular cytotoxicity (ADCC) is based on the principle that soluble complexes can compete with target cell-bound antibody for receptors (FcR) on cytotoxic lymphocytes. The objective of this study was to define a cytotoxicity system for the determination of soluble immune complexes in the sera of patients with inflammatory bowel disease (IBD). For this purpose, the conditions under which soluble complexes of rat serum albumin (RSA) and rabbit anti-RSA inhibited human K-cell mediated lysis of sensitized Chang cells were examined, on the assumption that the behaviour in the system of circulating immune complexes putatively present in inflammatory bowel disease, is similar to that of artificial immune complexes. Inhibition of ADCC by a standard amount of artificial complex in different normal human sera was relatively uniform provided that the final concentration of the latter did not exceed 10% of the culture medium. In the absence of extraneous complexes, the effect of both normal and IBD sera on ADCC varied widely. Differential inhibition of ADCC by sera from patients with IBD and normal
INTRODUCTION
Circulating immune complexes have been found in a number of pathological conditions. They may be detected by several techniques based on physiochemical properties of aggregated immunoglobulins or artificially prepared immune complexes, such as large molecular weight (Kunkel, Muller-Eberhard, Fudenberg & Tomasi, 1961), complement fixation (Agnello, Winchester & Kunkel, 1970; Nydegger, Lambert, Gerber & Miescher, 1974; Johnson, Mowbray & Porter, 1975; Lurhuma, Cambiaso, Masson & Heremans, 1976; Hay, Nineham & Roitt, 1976), cryoprecipitation (Meltzer & Franklin, 1966), susceptibility to phagocytosis by macrophages (Onyewotu, Holborow
* Present address: Department of Surgery, Wythenshawe Hospital, Manchester. Correspondence: Dr Michael Moore, Immunology Department, Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester M20 9BX. 0019-2805/79/0600-0467 $02.00 (O 1979 Blackwell Scientific Publications
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& Johnson, 1974), exposure of Fc determinants which are then detected by rheumatoid factor (Winchester, Kunkel & Agnello, 1971) and competitive inhibition of the Fc receptor of K cells (Jewell & Maclennan, 1973; Theofilopoulos, Wilson & Dixon, 1976; Panayi, Poston & Corrigall, 1977). The presence of immune complexes in pathological sera examined by any of these methods may only be inferred. Their actual presence would require a direct demonstration of antigen and antibody and of their combination. In very few instances is the nature of the antigen known. Techniques upon which the presence of immune complexes is merely inferred require caution in interpretation since sera may have indirect effects on the assay attributable to factors other than immune complexes. One such pervasive possibility is aggregated (non-complexed) IgG. Detection of immune complexes by inhibition of antibody-mediated lymphocyte cytotoxicity is based on the principle that the soluble complexes compete with target cell-bound antibody for Fc receptors on cytotoxic lymphocytes. In this study we have examined some of the variables associated with the detection of immune complexes in pathological sera by an exemplification of this method, viz, the Chang system of MacLennan (1972). Sera from patients with inflammatory bowel disorders were studied as there is increasing, though in terms of the above qualification, indirect evidence, implicating circulating immune complexes in the disease process.
Artificial immune complexes Antisera (anti-RSA) were prepared in rabbits by hyperimmunization with rat serum albumin (RSA) as previously described (Hilton, Moore & Howat, 1978). Immune complexes were prepared by admixture of RSA and anti-RSA over a wide range of antigen-antibody dilutions. To determine the relationship of inhibition of cytotoxicity to the degree of antigen binding, RSA was iodinated by the iodine monochloride technique. Different quantities of 125I-labelled RSA were added to standard amounts of antibody, followed by precipitation with 50% saturated ammonium sulphate, essentially as described by MacLennan (1972) for human serum albumin. Heat aggregated human IgG (HAGG) Human IgG was obtained from the serum of a patient with an IgG myeloma. IgG was precipitated by the addition of (NH4)2S04 to a final concentration of 333% (v/v), washed, resuspended and extensively dialysed against phosphate buffered saline (PBS). It was aggregated by heating at 63° for 15 min at a concentration of 21 mg/ml. Storage was at -70°.
PATIENTS AND METHODS
Effector cells Heparinized blood samples (20-100 ml) were obtained from the same donor (JMTH). Leucocyte preparations containing a high percentage of lymphocytes were separated by Ficoll-Triosil gradient centrifugation (Potter & Moore, 1975) and washed prior to test. Differential cell counts on seventeen stained smears of the washed interfacial preparations gave values of 79 + 4% lymphocytes, 8 + 3% polymorphonuclear leucocytes and 14 + 3% monocytes.
Patients The patients with inflammatory bowel disease (IBD) comprised twenty-three ulcerative colitis and twentyeight with Crohn's disease. They demonstrated all clinical grades of severity of disease from full remission to severe relapse and in each case diagnosis and extent of disease was confirmed by radiological and histological examination (Truelove & Witts, 1955; de Dombal, Burton, Clamp & Goligher, 1974). -Mean ages of male and female patients were 41 + 12 and 41 + 15 years, respectively. Control sera were obtained from fifty-two healthy subjects (forty-one males, mean age 43+ 16 years; eleven females, mean age 36 + 14 years), including patients with minor surgical conditions (obtained pretreatment) blood donors and laboratory staff.
Cytotoxicity test procedure Target cell damage was assessed by release of [5I Cr]chromate from Chang cells pre-labelled with this isotope, essentially as described by MacLennan (1972). Briefly, cultures were set up in triplicate in LP3 tubes, each containing lymphoid cells (5 x 105 in 200 p1), target cells (5 x 103 in 200 p1) and rabbit anti-Chang antibody (200 p1; 1 x 10- 5 dilution). All dilutions were made in Eagle's MEM supplemented with 10% foetal calf serum (Biocult Laboratories, Scotland) plus antibiotics. The same batch, which was most supportive of ADCC, was used throughout the study. Preparatory to the tests on pathological sera, preformed artificial complexes of RSA-anti-RSA suspended in either PBS or normal human sera were added in 200 p1 aliquots so as to give a total volume of each culture of 0 8 ml.
Inhibition of antibody-dependent cellular cytotoxicity Control tubes without effector cells or sensitizing antiserum, or both, were included in each experiment and maximum isotope release was determined by replacement of effectors with 200 p1 Triton (diluted 1/50). In experiments where the inhibitory capacity of pathological and normal human sera was compared further control tubes containing 500 pg HAGG in 200 pl were included to determine the maximum degree of inhibition attainable in the assay. Incubation was for 18-20 h at 370 in an atmosphere of 5% CO2 in air. Termination was by simultaneous centrifugation (400 g) of each tube for 10 min at 40. Aliquots (200 pl) of each supernatant and the residual supernatant and pellet were counted in a Searle Nuclear Chicago Automatic Counter (Model 1185). Expression of cytotoxicity data Percentage cytotoxicity (5I Cr release) was calculated from the formula: Radioactivity in supernatant removed ( x 4) 100 Total radioactivity in supernatant and residue
where all tubes were corrected for background radioactivity. To allow for variable degrees of spontaneous isotope release from target cells in different experiments, the results were expressed as a cytotoxic index and calculated from the formula: CRTest-CRSR 100
CRmax -CRSR where CRtest, CRmax and CRSR represent "Cr release in the test (percentage cytotoxicity), in the presence of Triton and in the absence ofeffector cells and antibody respectively. The percentage inhibition of cytotoxicity was calculated from the formula: CRTest-X 100 CRTest- GQO where CRTest = percentage 5'Cr release in the absence ofany inhibitory factor, X = percentage I 'Cr release in the presence of serum or artificial immune complex and G500 = percentage 5'Cr release in the presence of 500 pg HAGG. In experiments with artificial immune complexes, HAGG was not included and in the above formula this factor was replaced by the percent ' Cr release in the presence of effector cells but in the absence of sensitiz-
469
ing antibody. This value was normally 5-10% higher than the spontaneous 'I Cr release (CRSR).
Immunoglobulin determination Immunoglobulin levels (IgG and IgM) in sera were measured by immunodiffusion using standard ICL Scientific immunoplates. Micro lyumphocyte cytotoxicity test For estimations ofHL-A antibodies, aliquots of serum were tested against a panel of eighteen lymphocytes in Terasaki plates according to standard tissue typing procedure, using rabbit serum as a source of complement.
PHA-induced lymphocyte transformation Purified peripheral blood mononuclear cells (from JMTH) were incubated at 370 in 0-2 ml RPM 1 1640 (Flow Laboratories) containing 10% of the serum under test and 0-2 pg purified PHA (Wellcome). Base line transformation indices were evaluated in the absence of PHA. After 72 h incubation, cultures were pulsed for 4 h with 1 pCi of [3H]-thymidine (specific activity 2-5 Ci/mmol) and then harvested in a Sam harvester (Cryotech Laboratories). Isotope incorporation was measured in a Packard Liquid Scintillation Spectrometer (model 3385). Statistics Data were statistically analysed by one- or two-factor ANOVA, or Kendal Rank Correlation Coefficient Test, as stated in the text.
RESULTS Inhibition of ADCC by artificial immune complexes in the absence of human serum Experiments were designed to determine the conditions under which immune complexes of rat serum albumin (RSA) and anti-rat serum albumin (antiRSA) were inhibitory for ADCC. Neither antigen nor antibody alone was inhibitory, but RSA: anti-RSA immune complexes were inhibitory over a wide range of antigen and antibody concentrations. Reference to Fig. 1 illustrates that there was an optimal inhibitory concentration of antigen for each antibody concentration and that inhibition was greatest at high antibody concentrations. At lower concentrations of antibody the amount of antigen required to produce maximum inhibition was less critical. The relationship between inhibition of ADCC and
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inhibition was most consistently acheived under conditions of antigen excess and declined steeply beyond equivalence into antibody excess, becoming indetectable at antigen concentrations below 0 03 jug/ml. A reduction in the efficacy of inhibition attributed to a moderate increase in antigen concentration in a closely similar system (MacLennan, 1972) was not observed in this experiment.
.,5 11
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Figure 1. Inhibition of antibody-dependent cellular cytotoxicity (ADCC) by immune complexes of rat serum albumin and rabbit anti-rat serum albumin (RSA: anti-RSA). (Culture conditions as described in Materials and Methods)
Inhibition of ADCC by artificial immune complexes in the presence of normal human serum Based on data derived from the radioimmunoprecipitation experiment, inhibition of ADCC by artificial immune complexes (RSA: rat anti-RSA) was quantified in the presence of several human sera. In the absence of immune complexes, normal human serum generally augmented ADCC over that observed in FBS-supplemented cultured media. Neither free antigen nor free antibody was inhibitory (Fig. 3); whereas the extent to which standard aliquots of immune complex were inhibitory depended on the concentration of human serum and was most variable at high concentrations. Under these latter conditions, the
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Figure 2. The relationship of inhibition of antibody-dependent cellular cytotoxicity (ADCC) to the degree of antigen (RSA) binding by antibody (anti-RSA). All tubes contained 1: 8 dilution ofanti-RSA. Percentage of radiolabelled antigen not precipitated by 50% saturated ammonium sulphate (free antigen), o, percentage inhibition of ADCC in relation to antigen concentration, *; cytotoxic index in relation to antigen concentration, *.
the degree of binding by antibody was determined by radioimmunoprecipitation. Antigen (RSA) concentration was varied while that of antibody (anti-RSA) remained constant (dilution, 1: 8). Figure 2 shows that
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Figure 3. Inhibition of antibody-dependent cellular cytotoxicity (ADCC) by immune complexes of RSA:anti-RSA in fresh normal human 'carrier' serum. Cytotoxic indices: in the presence of anti-Chang antibody, A; in the absence of antiChang antibody, e; when antigen (RSA; 30 yg) only was present, a; when antiserum (anti-RSA; 1:10) only was present, v; and when immune complexes were present, o; percentage inhibition by immune complexes in relation to 'carrier' serum concentration, *.
Inhibition ofantibody-dependent cellular cytotoxicity effe(ct of different sera on ADCC varied between the extr emes where inhibition was either total or minimal, whil1st at higher dilution (> 1/10) there was closer corr*espondence between different sera. Statistical ana lysis (two-factor ANOVA) confirmed what the grarphical data (Fig. 4) suggest viz. that the presence of Eserum significantly affects percentage inhibition by a standard amount of immune complex, F(3,,40) = 18 49, P < 5%O lymphocytes in the test panel, each of which were from IBD patients. Only one ofthese sera reacted with lymphocytes from the ADCC donor. Weak reactions (i.e. against one or two lymphocytes in the panel) were observed for four others, of which two were pathological and two were normal sera.
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0 20 40 60 80 -80 -60 -40 -20 Percentage inhibition of ADCC with respect to foetal bovine serum
Figure 5. Relative inhibition of ADCC by sera of (a) patients with inflammatory bowel disease (IBD) and (b) normal subjects expressed as a function of ADCC in the presence of a standard batch of foetal bovine serum (FBS). Negative values denote an increase in ADCC compared with the FBS standard, and positive values, a decrease. For explanation, see Results.
PHA-induced lymphocyte transformation in normal and pathological sera Sera from thirty-six out of fifty-two patients with IBD and five normal subjects were tested for their capacity to support PHA-induced transformation of effector lymphocytes in a single assay. Values for the PHAstimulated and unstimulated cultures in the presence
Inhibition ofantibody-dependent cellular cytotoxicity
473
Table 3. PHA-induced transformation of effector lymphocytes in the presence of normal and pathological sera
Gross counts + 1 SD
Patient group (No. tested) All subjects (36) IBD patients (31) Healthy controls (5) (Foetal bovine serum)
No. sera > 2 SD from mean
Unstimulated PHA-Stimulated Normal 446 + 358 469 + 371 299 + 208 2227
of the various sera are given in Table 3. Overall, there was no difference between the degree of lymphocyte transformation in IBD and normal sera. Ten IBD sera differed by more than 2 SD from the normal mean; two enhanced and eight gave diminished responses. In none of the latter, however, where transformation was reduced in comparison with normal sera, did the gross counts fall below those obtained in FBS. In the ADCC assay, neither of two enhancing sera differed significantly from the normal mean, whereas four out of eight sera in which PHA-induced lymphocyte responses were reduced, were inhibitory. DISCUSSION
Inhibition experiments with artificially prepared soluble immune complexes (RSA: anti-RSA) in the absence of human serum were reproducible and showed general agreement with the earlier data of MacLennan (1972) using rat spleen cells as effectors in the ADCC assay. Indications that the assay might be useful in a clinical context were that inhibition was detectable over a wide range of antigen and antibody concentrations, and under optimal conditions, those of antigen excess could be detected to a concentration of 0 03 pg antigen/ml. The inhibition produced by a standard amount of artificial immune complex in the presence of normal human sera showed marked disparity between different sera at high concentrations. The ~effect of serum dilution was also complex in that, while more uniform than at high concentrations, the degree of inhibition varied for the same concentration of different sera. This would appear to be due either to non-specific interference with the assay by unspecified plasma components or the generation of further soluble immune complexes in the culture by the reaction of anti-rat serum albumin with human serum albumin. The latter
57,194 + 14,979 57,855 + 12,225 64,375 + 6,581 27,889
0 0 0
IBD
0 2(-) 2(+), 8(-) -
explanation would not necessarily be excluded by failure to detect cross-reactivity by immunodiffusion. These data indicated that a final dilution of 10% (v/v) was the highest permissible concentration at which pathological sera could be analysed for factors inhibitory of ADCC. The variable concentration effect ofdifferent normal human sera was also one of the factors which dictated the choice of a single batch of FBS as the reference standard against which to compare the effects of normal and pathological sera on ADCC. It is common experience that different batches of FBS, which contain little or no IgG, support different levels of lymphocyte-mediated cytotoxicity. The differences are presumably of a nutritional nature though the possibility that actively inhibitory material is involved, cannot be discounted. The batch used in the present study supported ADCC less effectively than the majority of normal human sera and it was therefore necessary to analyse the data in a way that this fact could be recognized. This aspect of experimental design amplifies a major problem in the interpretation of the test; that quantification of cytotoxicity in human sera is a function of several variables, including the differential capacity of individual sera to provide support for cytotoxic cell-to-cell interactions, and may oppose the effect of factors actively inhibitory of ADCC. This is a feature of all sera, normal and pathological, and it is responsible for the difficulty in establishing a base-line with which to compare the activity of the two groups. It is for this reason that the histogram mode of presentation has been adopted and quoted median values are based upon non-parametric statistics. The effect of normal and IBD sera on ADCC revealed highly significant differences in all but one of six comparable tests. There was no difference, however, between the activity of sera of patients with ulcerative colitis and Crohn's disease. These data as
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such did not determine whether the effect of IBD patients sera was attributable to factors inhibitory of ADCC or to a deficit in support. To investigate the latter possibility, sera from IBD patients and normal subjects were tested for their capacity to support PHAinduced transformation of effector lymphocytes. The majority of IBD sera sustained lymphocyte transformation at least as well as normal sera suggesting that inhibition of ADCC was not due to some nutritional or other serum deficit. Moreover, ofthe eight IBD sera which were most inhibitory in the ADCC assays, four gave rise to diminished transformation, although the latter still exceeded that in foetal bovine serum. We interpret these observations to indicate that inhibition of ADCC by these sera is more likely to be due to an active phenomenon, than to a deficit in support. The possibility that HL-A antibodies, arising through pregnancy and/or transfusion in the sera of IBD patients, might lyse the effector lymphocytes was also eliminated as an artefact of the test system. Our data are consistent with the view that the inhibition of ADCC by IBD sera is an active phenomenon and may be attributed to aggregates or immune complexes of IgG. Although a large series of patients was studied, the incidence of inhibitory sera is difficult to define on account of the variation between normal individuals. This may be due to the transient presence of immune complexes in the sera of healthy subjects as a result of, for example, the enteric uptake of food antigens or subclinical infections (Lurhuma et al., 1976). Even so, it is evident that, taken as a group, the sera of some IBD patients contained inhibitory factors outside the normal range and may be of potential pathological significance. The absence of a correlation between inhibitory acitivity and total serum IgG indicates that inhibition is unlikely to be due to aggregates, and is probably attributable to immune complexes. Other factors have important implications for the detection of circulating immune complexes by this technique. Panayi et al. (1977) stressed the importance of the ratio of mononuclear cells to target cells. The presence of polymorphonuclear leucocytes, while not active per se (MacLennan, 1972) might reduce the efficiency of the system by competitive binding of phagocytosis of the material via their Fc receptors. In the present study the number of polymorphs being separated by a standard procedure from the same donor, was relatively constant in each experiment, but no attempt was made to eliminate them. There is also the possibility that levels of ADCC
may be augmented by the presence of complement. All sera used in this study were unheated, so as to minimize the risk of aggregate formation. Complement is not a requirement for ADCC and complement-mediated lysis of Chang targets was precluded by the use of a high dilution of anti-Chang serum. Enhancement of ADCC by complement, however, in the absence of concomitant antibody-complement mediated cytotoxicity, has been reported in other systems (Rouse, Grewal & Babiuk, 1977). While this may constitute another variable in the ADCC system, it does not entirely account for the differential support of most human sera and foetal bovine serum, since the latter was also unheated. Immune complexes in inflammatory bowel disease have been demonstrated by other techniques; an anticomplementary assay (Johnson et al., 1975); precipitation with Clq (Doe, Booth & Brown, 1973); and inhibition of complement-dependent lymphocytic rosette formation (Ezer & Hayward, 1974). Although differences are reported in the incidence and distribution of immune complexes in Crohn's disease and ulcerative colitis there is general concordance between data derived from the various tests. Among those patients in whom immune complexes were detectable, there was a positive correlation with the severity of disease as determined by the anticomplementary test (Hodgson, Potter & Jewell, 1977a; Nielsen, Binder, Daugharty & Svehag, 1978a) and inhibition of ADCC (Jewell & MacLennan, 1973). Immune complexes were found more frequently in the presence of active intestinal disease and in particular when extra-intestinal manifestations were evident. This correlation with clinical status could account, in part, for the lower incidence (I 5%) of IBD sera containing immune complexes observed in the present study, in comparison with some other estimates (30%) (Jewell & MacLennan, 1973; Hodgson, et al., 1977a), since only a small minority of our patients had fulminating disease. Even so, it would appear that immune complexes are indetectable in the majority of IBD patients sera regardless of the technique employed, and the role of immune complexes in the pathogenesis of these diseases must therefore be interpreted with this awareness. Their presence in the circulation may be secondary to mucosal damage, while their local formation and deposition within the mucosa itself could be responsible for the initiation and maintenance of the inflammatory changes. The appearance of C3 breakdown products in the serum (Teisburg & Gjone, 1975) and hypercatabolism of C3 (Hodgson, Potter &
Inhibition of antibody-dependent cellular cytotoxicity Jewell, 1977b; Nielsen, Peterson & Svehag, 1978b) indicating complement activation in patients with IBD would support the latter hypothesis, but as yet the nature of the antigen(s) in the immune complex is unknown. ACKNOWLEDGMENTS
We thank Mr P. F. Schofield FRCS for access to his patients and Dr Michael Palmer for statistical analyses. HL-A antibodies were kindly determined by Dr P. Klouda, Department of Medical Genetics, St. Mary's Hospital, Manchester and immunoglobulin estimations by Dr. E. Gowland, Department of Chemical Pathology, Withington Hospital, Manchester. Normal sera were obtained by courtesy of the Regional Blood Transfusion Service, Picadilly, Manchester. This work was supported by the Medical Research Council and the Cancer Research Campaign. JMTH was in receipt of an MRC Clinical Fellowship.
REFERENCES AGNELLO V., WINCHESTER R.J. & KUNKEL H.G. (1970) Precipitin reactions of the C I q component ofcomplement with aggregated y -globulin and immune complexes in gel
diffusion. Immunology, 19,909. DE DOMBAL F.T., BURTON I.L., CLAMP S.E. & GOLIGHER J.C. (1974) Short-term course and prognosis of Crohn's
disease. Gut, 15, 435. DOE W.F., BOOTH C.C. & BROWN D.L. (1973) Evidence for complement-binding immune complexes in adult coeliac disease, Crohn's disease and ulcerative colitis. Lancet, i, 402. EZER G. & HAYWARD A.R. (1974) Inhibition ofcomplementdependent lymphocyte rosette formation: a possible test for activated complement products. Europ. J. Immunol. 4, 148. HAY F.C., NINEHAM L.J. & RoITT I.M. (1976) Routine assay for the detection of immune complexes of known immunoglobulin class using solid phase Clq. Clin. exp. Immunol. 24, 396. HILTON, A.M., MOORE, M. & HOWAT, J.M.T. (1978) Detection of circulating anticomplementary factors in chronic lung diseases. Clin. exp. Immunol. 31, 237. HODGSON H.J.F., POTTER B.J. & JEWELL D.P. (1977a) Immune complexes in ulcerative colitis and Crohn's disease. Clin. exp. Immunol. 29, 187. HODGSON H.J.F., POTTER B.J. & JEWELL D.P.(1977b) C3 metabolism in ulcerative colitis and Chrohn's disease. Clin. exp. Immunol. 28, 490. JOHNSON A.H., MOWBRAY J.F. & PORTER K.A. (1975) Detec-
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tion of circulating immune complexes in pathological human sera. Lancet, i, 762. JEWELL D.P. & MAcLENNAN I.C.M. (1973) Circulating immune complexes in inflammatory bowel disease. Clin. exp. Immunol. 14, 219. KUNKEL H.G., MuLLER-EBERHARD H.J., FUDENBERG H.H. & TOMASI T.B. (1961) Gamma-Globulin complexes in rheumatoid arthritis and certain other conditions. J. clin. Invest. 40, 117. LURHUMA A.Z., CAMBIASO C.C., MASSON P.L. & HEREMANS J.F. (1976) Detection of circulating antigen-antibody complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatoid factor of Clq. Clin. exp. Immunol. 25, 212. MAcLENNAN I.C.M. (1972) Competition for receptors for immunoglobulin on cytotoxic lymphocytes. Clin. exp. Immunol. 10, 275. MELTZER M. & FRANKLIN E.C. (1966) Cryoglobulinaemia: a study of twenty-nine patients. Am. J. Med. 40, 828. NIELSEN H., BINDER V., DAUGHARTY H. & SVEHAG S.-E. (1978a) Circulating immune complexes in ulcerative colitis I. Correlation to disease activity. Clin. exp. Immunol. 31, 72. NIELSEN H., PETERSEN P.H. & SVEHAG S.-E. (1978b) Circulating immune complexes in ulcerative colitis II. Correlation with serum protein concentrations and complement conversion products. Clin. exp. Immunol. 31, 81. NYDEGGER U.E., LAMBERT P.H., GERBER H. & MIESCHER P.A. (1974) Circulating immune complexes in the serum of systemic lupus erythematosus and in carriers of hepatitis B antigen. Quantitation by binding to radiolabelled Clq. J. clin. Invest. 54, 297. ONYEWOTU I.I., HOLBOROW E.J. & JOHNSON G.D. (1974) Detection and radioassay of soluble circulating immune complexes using guinea-pig perioneal exudate cells. Nature (Lond.) 248, 156. PANAYI G.S., POSTON R.N. & CORRIGALL V. (1977) Inhibition of antibody-mediated. lymphocyte cytotoxicity by aggregated human immunoglobulin G. Ann. Rheum. Dis. 36, (Suppl.) 59. POTTER M.R. & MOORE M. (1975) PHA stimulation of separated human lymphocyte populations. Clin. exp. Immuno/. 21, 456. ROUSE B.T., GREWAL A.S. & BABIUK L.A. (1977) Complement enhances anti-viral antibody-dependent cell cytotoxicity. Nature (Lond.) 266, 456. TEISBURG P.A. & GJoNE E. (1975) Humoral immune system activity in inflammatory bowel disease. Scand. J. Gastroent. 10, 545. THEOFILOPOULOS A.N., WILSON C.B. & DIXON F.J. (1976) The Raji cell radioimmune assay for detecting immune complexes in human sera. J. clin. Invest. 57, 169. TRUELOVE S.C. & WITTS L.J. (1955) Cortisone in ulcerative colitis: final report on a therapeutic trial. Br. Med. J. ii, 1041. WINCHESTER K.J., KUNKEL H.G. & AGNELLO V. (1971) Occurrence of gammaglobulin complexes in serum and joint fluids of rheumatoid arthritis patients. Use of monoclonal rheumatoid factors as reagents for their demonstration. J. exp. Med. 134,286.
Inhibition of antibody-dependent cellular cytotoxicity by artificial immune complexes and pathological sera
J. M. T. HOWAT*, M. MOORE, ANGELA M. HILTON& I. KIMBERImmunology Department, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 18 October 1978: acceptedfor publication 6 November 1978
subjects was thus expressed as a function of ADCC in a standard batch of foetal bovine serum (FBS). Under these conditions differences between pathological (n= 51) and normal (n= 52) sera were highly significant (P < 0 001), which could not be explained by the presence in the patients' sera of H L-A antibodies reactive with the effector cells, nor by a deficit in nutritional support of ADCC. The absence of a correlation between inhibition of ADCC and total serum IgG or IgM inferred that inhibition was attributable to immune complexes in the IBD sera. The limitations of this assay for assessment of the incidence of immune complexes in pathological sera are discussed.
Summary. Detection of immune complexes by inhibition of antibody-dependent cellular cytotoxicity (ADCC) is based on the principle that soluble complexes can compete with target cell-bound antibody for receptors (FcR) on cytotoxic lymphocytes. The objective of this study was to define a cytotoxicity system for the determination of soluble immune complexes in the sera of patients with inflammatory bowel disease (IBD). For this purpose, the conditions under which soluble complexes of rat serum albumin (RSA) and rabbit anti-RSA inhibited human K-cell mediated lysis of sensitized Chang cells were examined, on the assumption that the behaviour in the system of circulating immune complexes putatively present in inflammatory bowel disease, is similar to that of artificial immune complexes. Inhibition of ADCC by a standard amount of artificial complex in different normal human sera was relatively uniform provided that the final concentration of the latter did not exceed 10% of the culture medium. In the absence of extraneous complexes, the effect of both normal and IBD sera on ADCC varied widely. Differential inhibition of ADCC by sera from patients with IBD and normal
INTRODUCTION
Circulating immune complexes have been found in a number of pathological conditions. They may be detected by several techniques based on physiochemical properties of aggregated immunoglobulins or artificially prepared immune complexes, such as large molecular weight (Kunkel, Muller-Eberhard, Fudenberg & Tomasi, 1961), complement fixation (Agnello, Winchester & Kunkel, 1970; Nydegger, Lambert, Gerber & Miescher, 1974; Johnson, Mowbray & Porter, 1975; Lurhuma, Cambiaso, Masson & Heremans, 1976; Hay, Nineham & Roitt, 1976), cryoprecipitation (Meltzer & Franklin, 1966), susceptibility to phagocytosis by macrophages (Onyewotu, Holborow
* Present address: Department of Surgery, Wythenshawe Hospital, Manchester. Correspondence: Dr Michael Moore, Immunology Department, Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester M20 9BX. 0019-2805/79/0600-0467 $02.00 (O 1979 Blackwell Scientific Publications
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& Johnson, 1974), exposure of Fc determinants which are then detected by rheumatoid factor (Winchester, Kunkel & Agnello, 1971) and competitive inhibition of the Fc receptor of K cells (Jewell & Maclennan, 1973; Theofilopoulos, Wilson & Dixon, 1976; Panayi, Poston & Corrigall, 1977). The presence of immune complexes in pathological sera examined by any of these methods may only be inferred. Their actual presence would require a direct demonstration of antigen and antibody and of their combination. In very few instances is the nature of the antigen known. Techniques upon which the presence of immune complexes is merely inferred require caution in interpretation since sera may have indirect effects on the assay attributable to factors other than immune complexes. One such pervasive possibility is aggregated (non-complexed) IgG. Detection of immune complexes by inhibition of antibody-mediated lymphocyte cytotoxicity is based on the principle that the soluble complexes compete with target cell-bound antibody for Fc receptors on cytotoxic lymphocytes. In this study we have examined some of the variables associated with the detection of immune complexes in pathological sera by an exemplification of this method, viz, the Chang system of MacLennan (1972). Sera from patients with inflammatory bowel disorders were studied as there is increasing, though in terms of the above qualification, indirect evidence, implicating circulating immune complexes in the disease process.
Artificial immune complexes Antisera (anti-RSA) were prepared in rabbits by hyperimmunization with rat serum albumin (RSA) as previously described (Hilton, Moore & Howat, 1978). Immune complexes were prepared by admixture of RSA and anti-RSA over a wide range of antigen-antibody dilutions. To determine the relationship of inhibition of cytotoxicity to the degree of antigen binding, RSA was iodinated by the iodine monochloride technique. Different quantities of 125I-labelled RSA were added to standard amounts of antibody, followed by precipitation with 50% saturated ammonium sulphate, essentially as described by MacLennan (1972) for human serum albumin. Heat aggregated human IgG (HAGG) Human IgG was obtained from the serum of a patient with an IgG myeloma. IgG was precipitated by the addition of (NH4)2S04 to a final concentration of 333% (v/v), washed, resuspended and extensively dialysed against phosphate buffered saline (PBS). It was aggregated by heating at 63° for 15 min at a concentration of 21 mg/ml. Storage was at -70°.
PATIENTS AND METHODS
Effector cells Heparinized blood samples (20-100 ml) were obtained from the same donor (JMTH). Leucocyte preparations containing a high percentage of lymphocytes were separated by Ficoll-Triosil gradient centrifugation (Potter & Moore, 1975) and washed prior to test. Differential cell counts on seventeen stained smears of the washed interfacial preparations gave values of 79 + 4% lymphocytes, 8 + 3% polymorphonuclear leucocytes and 14 + 3% monocytes.
Patients The patients with inflammatory bowel disease (IBD) comprised twenty-three ulcerative colitis and twentyeight with Crohn's disease. They demonstrated all clinical grades of severity of disease from full remission to severe relapse and in each case diagnosis and extent of disease was confirmed by radiological and histological examination (Truelove & Witts, 1955; de Dombal, Burton, Clamp & Goligher, 1974). -Mean ages of male and female patients were 41 + 12 and 41 + 15 years, respectively. Control sera were obtained from fifty-two healthy subjects (forty-one males, mean age 43+ 16 years; eleven females, mean age 36 + 14 years), including patients with minor surgical conditions (obtained pretreatment) blood donors and laboratory staff.
Cytotoxicity test procedure Target cell damage was assessed by release of [5I Cr]chromate from Chang cells pre-labelled with this isotope, essentially as described by MacLennan (1972). Briefly, cultures were set up in triplicate in LP3 tubes, each containing lymphoid cells (5 x 105 in 200 p1), target cells (5 x 103 in 200 p1) and rabbit anti-Chang antibody (200 p1; 1 x 10- 5 dilution). All dilutions were made in Eagle's MEM supplemented with 10% foetal calf serum (Biocult Laboratories, Scotland) plus antibiotics. The same batch, which was most supportive of ADCC, was used throughout the study. Preparatory to the tests on pathological sera, preformed artificial complexes of RSA-anti-RSA suspended in either PBS or normal human sera were added in 200 p1 aliquots so as to give a total volume of each culture of 0 8 ml.
Inhibition of antibody-dependent cellular cytotoxicity Control tubes without effector cells or sensitizing antiserum, or both, were included in each experiment and maximum isotope release was determined by replacement of effectors with 200 p1 Triton (diluted 1/50). In experiments where the inhibitory capacity of pathological and normal human sera was compared further control tubes containing 500 pg HAGG in 200 pl were included to determine the maximum degree of inhibition attainable in the assay. Incubation was for 18-20 h at 370 in an atmosphere of 5% CO2 in air. Termination was by simultaneous centrifugation (400 g) of each tube for 10 min at 40. Aliquots (200 pl) of each supernatant and the residual supernatant and pellet were counted in a Searle Nuclear Chicago Automatic Counter (Model 1185). Expression of cytotoxicity data Percentage cytotoxicity (5I Cr release) was calculated from the formula: Radioactivity in supernatant removed ( x 4) 100 Total radioactivity in supernatant and residue
where all tubes were corrected for background radioactivity. To allow for variable degrees of spontaneous isotope release from target cells in different experiments, the results were expressed as a cytotoxic index and calculated from the formula: CRTest-CRSR 100
CRmax -CRSR where CRtest, CRmax and CRSR represent "Cr release in the test (percentage cytotoxicity), in the presence of Triton and in the absence ofeffector cells and antibody respectively. The percentage inhibition of cytotoxicity was calculated from the formula: CRTest-X 100 CRTest- GQO where CRTest = percentage 5'Cr release in the absence ofany inhibitory factor, X = percentage I 'Cr release in the presence of serum or artificial immune complex and G500 = percentage 5'Cr release in the presence of 500 pg HAGG. In experiments with artificial immune complexes, HAGG was not included and in the above formula this factor was replaced by the percent ' Cr release in the presence of effector cells but in the absence of sensitiz-
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ing antibody. This value was normally 5-10% higher than the spontaneous 'I Cr release (CRSR).
Immunoglobulin determination Immunoglobulin levels (IgG and IgM) in sera were measured by immunodiffusion using standard ICL Scientific immunoplates. Micro lyumphocyte cytotoxicity test For estimations ofHL-A antibodies, aliquots of serum were tested against a panel of eighteen lymphocytes in Terasaki plates according to standard tissue typing procedure, using rabbit serum as a source of complement.
PHA-induced lymphocyte transformation Purified peripheral blood mononuclear cells (from JMTH) were incubated at 370 in 0-2 ml RPM 1 1640 (Flow Laboratories) containing 10% of the serum under test and 0-2 pg purified PHA (Wellcome). Base line transformation indices were evaluated in the absence of PHA. After 72 h incubation, cultures were pulsed for 4 h with 1 pCi of [3H]-thymidine (specific activity 2-5 Ci/mmol) and then harvested in a Sam harvester (Cryotech Laboratories). Isotope incorporation was measured in a Packard Liquid Scintillation Spectrometer (model 3385). Statistics Data were statistically analysed by one- or two-factor ANOVA, or Kendal Rank Correlation Coefficient Test, as stated in the text.
RESULTS Inhibition of ADCC by artificial immune complexes in the absence of human serum Experiments were designed to determine the conditions under which immune complexes of rat serum albumin (RSA) and anti-rat serum albumin (antiRSA) were inhibitory for ADCC. Neither antigen nor antibody alone was inhibitory, but RSA: anti-RSA immune complexes were inhibitory over a wide range of antigen and antibody concentrations. Reference to Fig. 1 illustrates that there was an optimal inhibitory concentration of antigen for each antibody concentration and that inhibition was greatest at high antibody concentrations. At lower concentrations of antibody the amount of antigen required to produce maximum inhibition was less critical. The relationship between inhibition of ADCC and
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inhibition was most consistently acheived under conditions of antigen excess and declined steeply beyond equivalence into antibody excess, becoming indetectable at antigen concentrations below 0 03 jug/ml. A reduction in the efficacy of inhibition attributed to a moderate increase in antigen concentration in a closely similar system (MacLennan, 1972) was not observed in this experiment.
.,5 11
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Figure 1. Inhibition of antibody-dependent cellular cytotoxicity (ADCC) by immune complexes of rat serum albumin and rabbit anti-rat serum albumin (RSA: anti-RSA). (Culture conditions as described in Materials and Methods)
Inhibition of ADCC by artificial immune complexes in the presence of normal human serum Based on data derived from the radioimmunoprecipitation experiment, inhibition of ADCC by artificial immune complexes (RSA: rat anti-RSA) was quantified in the presence of several human sera. In the absence of immune complexes, normal human serum generally augmented ADCC over that observed in FBS-supplemented cultured media. Neither free antigen nor free antibody was inhibitory (Fig. 3); whereas the extent to which standard aliquots of immune complex were inhibitory depended on the concentration of human serum and was most variable at high concentrations. Under these latter conditions, the
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Figure 2. The relationship of inhibition of antibody-dependent cellular cytotoxicity (ADCC) to the degree of antigen (RSA) binding by antibody (anti-RSA). All tubes contained 1: 8 dilution ofanti-RSA. Percentage of radiolabelled antigen not precipitated by 50% saturated ammonium sulphate (free antigen), o, percentage inhibition of ADCC in relation to antigen concentration, *; cytotoxic index in relation to antigen concentration, *.
the degree of binding by antibody was determined by radioimmunoprecipitation. Antigen (RSA) concentration was varied while that of antibody (anti-RSA) remained constant (dilution, 1: 8). Figure 2 shows that
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Figure 3. Inhibition of antibody-dependent cellular cytotoxicity (ADCC) by immune complexes of RSA:anti-RSA in fresh normal human 'carrier' serum. Cytotoxic indices: in the presence of anti-Chang antibody, A; in the absence of antiChang antibody, e; when antigen (RSA; 30 yg) only was present, a; when antiserum (anti-RSA; 1:10) only was present, v; and when immune complexes were present, o; percentage inhibition by immune complexes in relation to 'carrier' serum concentration, *.
Inhibition ofantibody-dependent cellular cytotoxicity effe(ct of different sera on ADCC varied between the extr emes where inhibition was either total or minimal, whil1st at higher dilution (> 1/10) there was closer corr*espondence between different sera. Statistical ana lysis (two-factor ANOVA) confirmed what the grarphical data (Fig. 4) suggest viz. that the presence of Eserum significantly affects percentage inhibition by a standard amount of immune complex, F(3,,40) = 18 49, P < 5%O lymphocytes in the test panel, each of which were from IBD patients. Only one ofthese sera reacted with lymphocytes from the ADCC donor. Weak reactions (i.e. against one or two lymphocytes in the panel) were observed for four others, of which two were pathological and two were normal sera.
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0 20 40 60 80 -80 -60 -40 -20 Percentage inhibition of ADCC with respect to foetal bovine serum
Figure 5. Relative inhibition of ADCC by sera of (a) patients with inflammatory bowel disease (IBD) and (b) normal subjects expressed as a function of ADCC in the presence of a standard batch of foetal bovine serum (FBS). Negative values denote an increase in ADCC compared with the FBS standard, and positive values, a decrease. For explanation, see Results.
PHA-induced lymphocyte transformation in normal and pathological sera Sera from thirty-six out of fifty-two patients with IBD and five normal subjects were tested for their capacity to support PHA-induced transformation of effector lymphocytes in a single assay. Values for the PHAstimulated and unstimulated cultures in the presence
Inhibition ofantibody-dependent cellular cytotoxicity
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Table 3. PHA-induced transformation of effector lymphocytes in the presence of normal and pathological sera
Gross counts + 1 SD
Patient group (No. tested) All subjects (36) IBD patients (31) Healthy controls (5) (Foetal bovine serum)
No. sera > 2 SD from mean
Unstimulated PHA-Stimulated Normal 446 + 358 469 + 371 299 + 208 2227
of the various sera are given in Table 3. Overall, there was no difference between the degree of lymphocyte transformation in IBD and normal sera. Ten IBD sera differed by more than 2 SD from the normal mean; two enhanced and eight gave diminished responses. In none of the latter, however, where transformation was reduced in comparison with normal sera, did the gross counts fall below those obtained in FBS. In the ADCC assay, neither of two enhancing sera differed significantly from the normal mean, whereas four out of eight sera in which PHA-induced lymphocyte responses were reduced, were inhibitory. DISCUSSION
Inhibition experiments with artificially prepared soluble immune complexes (RSA: anti-RSA) in the absence of human serum were reproducible and showed general agreement with the earlier data of MacLennan (1972) using rat spleen cells as effectors in the ADCC assay. Indications that the assay might be useful in a clinical context were that inhibition was detectable over a wide range of antigen and antibody concentrations, and under optimal conditions, those of antigen excess could be detected to a concentration of 0 03 pg antigen/ml. The inhibition produced by a standard amount of artificial immune complex in the presence of normal human sera showed marked disparity between different sera at high concentrations. The ~effect of serum dilution was also complex in that, while more uniform than at high concentrations, the degree of inhibition varied for the same concentration of different sera. This would appear to be due either to non-specific interference with the assay by unspecified plasma components or the generation of further soluble immune complexes in the culture by the reaction of anti-rat serum albumin with human serum albumin. The latter
57,194 + 14,979 57,855 + 12,225 64,375 + 6,581 27,889
0 0 0
IBD
0 2(-) 2(+), 8(-) -
explanation would not necessarily be excluded by failure to detect cross-reactivity by immunodiffusion. These data indicated that a final dilution of 10% (v/v) was the highest permissible concentration at which pathological sera could be analysed for factors inhibitory of ADCC. The variable concentration effect ofdifferent normal human sera was also one of the factors which dictated the choice of a single batch of FBS as the reference standard against which to compare the effects of normal and pathological sera on ADCC. It is common experience that different batches of FBS, which contain little or no IgG, support different levels of lymphocyte-mediated cytotoxicity. The differences are presumably of a nutritional nature though the possibility that actively inhibitory material is involved, cannot be discounted. The batch used in the present study supported ADCC less effectively than the majority of normal human sera and it was therefore necessary to analyse the data in a way that this fact could be recognized. This aspect of experimental design amplifies a major problem in the interpretation of the test; that quantification of cytotoxicity in human sera is a function of several variables, including the differential capacity of individual sera to provide support for cytotoxic cell-to-cell interactions, and may oppose the effect of factors actively inhibitory of ADCC. This is a feature of all sera, normal and pathological, and it is responsible for the difficulty in establishing a base-line with which to compare the activity of the two groups. It is for this reason that the histogram mode of presentation has been adopted and quoted median values are based upon non-parametric statistics. The effect of normal and IBD sera on ADCC revealed highly significant differences in all but one of six comparable tests. There was no difference, however, between the activity of sera of patients with ulcerative colitis and Crohn's disease. These data as
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such did not determine whether the effect of IBD patients sera was attributable to factors inhibitory of ADCC or to a deficit in support. To investigate the latter possibility, sera from IBD patients and normal subjects were tested for their capacity to support PHAinduced transformation of effector lymphocytes. The majority of IBD sera sustained lymphocyte transformation at least as well as normal sera suggesting that inhibition of ADCC was not due to some nutritional or other serum deficit. Moreover, ofthe eight IBD sera which were most inhibitory in the ADCC assays, four gave rise to diminished transformation, although the latter still exceeded that in foetal bovine serum. We interpret these observations to indicate that inhibition of ADCC by these sera is more likely to be due to an active phenomenon, than to a deficit in support. The possibility that HL-A antibodies, arising through pregnancy and/or transfusion in the sera of IBD patients, might lyse the effector lymphocytes was also eliminated as an artefact of the test system. Our data are consistent with the view that the inhibition of ADCC by IBD sera is an active phenomenon and may be attributed to aggregates or immune complexes of IgG. Although a large series of patients was studied, the incidence of inhibitory sera is difficult to define on account of the variation between normal individuals. This may be due to the transient presence of immune complexes in the sera of healthy subjects as a result of, for example, the enteric uptake of food antigens or subclinical infections (Lurhuma et al., 1976). Even so, it is evident that, taken as a group, the sera of some IBD patients contained inhibitory factors outside the normal range and may be of potential pathological significance. The absence of a correlation between inhibitory acitivity and total serum IgG indicates that inhibition is unlikely to be due to aggregates, and is probably attributable to immune complexes. Other factors have important implications for the detection of circulating immune complexes by this technique. Panayi et al. (1977) stressed the importance of the ratio of mononuclear cells to target cells. The presence of polymorphonuclear leucocytes, while not active per se (MacLennan, 1972) might reduce the efficiency of the system by competitive binding of phagocytosis of the material via their Fc receptors. In the present study the number of polymorphs being separated by a standard procedure from the same donor, was relatively constant in each experiment, but no attempt was made to eliminate them. There is also the possibility that levels of ADCC
may be augmented by the presence of complement. All sera used in this study were unheated, so as to minimize the risk of aggregate formation. Complement is not a requirement for ADCC and complement-mediated lysis of Chang targets was precluded by the use of a high dilution of anti-Chang serum. Enhancement of ADCC by complement, however, in the absence of concomitant antibody-complement mediated cytotoxicity, has been reported in other systems (Rouse, Grewal & Babiuk, 1977). While this may constitute another variable in the ADCC system, it does not entirely account for the differential support of most human sera and foetal bovine serum, since the latter was also unheated. Immune complexes in inflammatory bowel disease have been demonstrated by other techniques; an anticomplementary assay (Johnson et al., 1975); precipitation with Clq (Doe, Booth & Brown, 1973); and inhibition of complement-dependent lymphocytic rosette formation (Ezer & Hayward, 1974). Although differences are reported in the incidence and distribution of immune complexes in Crohn's disease and ulcerative colitis there is general concordance between data derived from the various tests. Among those patients in whom immune complexes were detectable, there was a positive correlation with the severity of disease as determined by the anticomplementary test (Hodgson, Potter & Jewell, 1977a; Nielsen, Binder, Daugharty & Svehag, 1978a) and inhibition of ADCC (Jewell & MacLennan, 1973). Immune complexes were found more frequently in the presence of active intestinal disease and in particular when extra-intestinal manifestations were evident. This correlation with clinical status could account, in part, for the lower incidence (I 5%) of IBD sera containing immune complexes observed in the present study, in comparison with some other estimates (30%) (Jewell & MacLennan, 1973; Hodgson, et al., 1977a), since only a small minority of our patients had fulminating disease. Even so, it would appear that immune complexes are indetectable in the majority of IBD patients sera regardless of the technique employed, and the role of immune complexes in the pathogenesis of these diseases must therefore be interpreted with this awareness. Their presence in the circulation may be secondary to mucosal damage, while their local formation and deposition within the mucosa itself could be responsible for the initiation and maintenance of the inflammatory changes. The appearance of C3 breakdown products in the serum (Teisburg & Gjone, 1975) and hypercatabolism of C3 (Hodgson, Potter &
Inhibition of antibody-dependent cellular cytotoxicity Jewell, 1977b; Nielsen, Peterson & Svehag, 1978b) indicating complement activation in patients with IBD would support the latter hypothesis, but as yet the nature of the antigen(s) in the immune complex is unknown. ACKNOWLEDGMENTS
We thank Mr P. F. Schofield FRCS for access to his patients and Dr Michael Palmer for statistical analyses. HL-A antibodies were kindly determined by Dr P. Klouda, Department of Medical Genetics, St. Mary's Hospital, Manchester and immunoglobulin estimations by Dr. E. Gowland, Department of Chemical Pathology, Withington Hospital, Manchester. Normal sera were obtained by courtesy of the Regional Blood Transfusion Service, Picadilly, Manchester. This work was supported by the Medical Research Council and the Cancer Research Campaign. JMTH was in receipt of an MRC Clinical Fellowship.
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