Immunological studies in ulcerative colitis. VI. Light microscopic studies with peroxidase conjugated antibodies

Cryostat sections of colon from germ-free rats were incubated with sera from patients with ulcerative colitis or healthy controls. With peroxidase-conjugated antibody from sheep to human immunoglobulin, a distinct staining of goblet cell surface was observed with the patients' sera but not with the controls. The staining compares well with that obtained by indirect immunofluorescence. It could easily be distinguished from staining due to endogenous peroxidase.     

Immunological studies in ulcerative colitis. III. Incidence of antibodies to colon-antigen in ulcerative colitis and other gastro-intestinal diseases

The presence of anti-colon antibodies in sera from patients with ulcerative colitis was investigated with indirect haemagglutination and the fluorescent antibody procedure. The antigen was obtained from germ-free rats. There was a good correlation between the results obtained with both procedures.

Of a group of 101 ulcerative colitis patients, 56% were found to have a haemagglutination titre of ≥ 1:16. In a group of forty-five age- and sex-matched healthy controls or surgical cases 13% had such titres. This difference was statistically significant. In an additional control group of patients with bronchial asthma, the incidence of elevated titres was higher than in the healthy controls but lower than in ulcerative colitis. Of 109 patients with other gastro-intestinal disorders (chronic diarrhoeas of unknown aetiology, bacillary dysentery, Salmonella infections, cancer of colon and rectum, coeliac disease) only eight had a titre of ≥ 1:16. In fifteen South African patients with amoebic dysentery but of unknown clinical status, the incidence of elevated haemagglutination titres was also higher than in the healthy controls (33%). Of eighteen patients with regional enteritis, twelve (67%) had titres of 1:16 or higher. However, eleven out of these eighteen had lesions in both large and small intestine. Fluorescent antibody staining of rat colon sections confirmed these results.

In ulcerative colitis, the incidence of elevated titres (≥ 1:16) was independent of the patients' age but was significantly higher in females older than 25 years than in male patients of the same age. In patients younger than 25 years, this difference between the sexes was less marked and statistically not significant. Colectomy (including pancoloproctectomy) was without influence on antibody titres, which sometimes were found to be elevated as long as 2–10 years after surgery. There was no significant correlation between antibody titres and duration or severity of the disease, the extent of colonic involvement and the occurrence of extra-colonic manifestations.

     

Anti-colon antibody and lymphocytophilic antibody in ulcerative colitis.

The presence of anti-colon antibody in the sera from patients with ulcerative colitis was demonstrated by antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In addition, the high prevalence of lymphocytophilic antibody in the sera from patients with ulcerative colitis was obtained by fluorescence activated cell sorter (FACS) analysis. This lymphocytophilic antibody was absorbed by rat colon epithelial cells. Moreover the lymphocytes from ulcerative colitis showed lower binding capacity to this antibody, but acquired higher binding capacity after 20 hr incubation at 37 degrees C in vitro. These data suggest that ADCC may have some role in the pathogenesis of ulcerative colitis.     

Anti-human cytomegalovirus nuclear antigen antibodies of different immunoglobulin classes

IgG, IgM and IgA immunoglobulin classes of antibodies to human cytomegalovirus nuclear antigens (CMNA) were studied by the acid-fixed nuclear binding technique (AFNB) and combined anti-complement immunofluorescence (combined ACIF). In acute cases of infectious mononucleosis (IM) of human cytomegalovirus (HCMV) origin and in the so-called double virus infections (HCMV + Epstein-Barr virus), anti-CMNA IgM antibodies were detected. They were absent from both anti-HCMV positive sera of healthy donors and sera of patients suffering of IM caused by EBV used as controls. The presence of anti-CMNA IgM may thus serve as an additional evidence of acute HCMV infection. Non-complement-fixing IgA classes of the anti-CMNA antibodies were not found in some of the sera gathered during the acute phase of IM of EBV origin: in one fourth of the HCMV seropositive donors and in a number of late serum samples. But non-complement-fixing and complement-fixing anti-CMNA components of the IgG class were detected.     

A solid-phase radioassay for the detection of anti-thyroglobulin antibodies in different immunoglobulin classes.

A convenient technique suitable for the routine estimation of IgG and IgA anti-thyroglobulin antibodies has been devised. The assay involves the binding of anti-thyroglobulin to human thyroglobulin linked to the surface of plastic tubes; the amount of antibody bound is then determined by adding radiolabelled anti-human IgG or IgA. IgG antibody was raised in virtually all patients and significantly elevated levels of IgA antibody were also found in some patients. The test should prove helpful in diagnosis and provide quantitative evaluation of research studies.     

Measurement of anti-double-stranded DNA antibodies in major immunoglobulin classes.

A solid-phase radioimmunoassay for quantitating anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) in IgG, IgM and IgA classes has been devised. A distinct feature of the method is an application of polystyrene tubes coated with poly-L-lysine, through which dsDNA could be bound firmly to a solid phase. Studies on patients' sea as well as normal sera revealed that anti-dsDNA was not qualitatively but quantitatively characteristic of systemic lupus erythematosus (SLE) and that IgG anti-dsDNA levels correlated well with the disease activity.     

Neutrophil cytoplasmic antibodies (p-ANCA) in ulcerative colitis.

AIMS--To study ulcerative colitis associated neutrophil cytoplasmic antibodies (p-ANCA) in respect of class and subclass distribution, antigen specificity, and (sub)cellular localisation of the antigen(s) to which these antibodies are directed. METHODS--p-ANCA positivity was determined using the standard indirect immunofluorescence test (IIFT). The immunoglobulin (Ig) subclass distribution of p-ANCA was investigated using monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4. Intracellular antigen localisation studies were performed on (fractionated) neutrophils using antigen-specific antibodies. RESULTS--In contrast to vasculitis associated ANCA, ulcerative colitis p-ANCA are mainly of IgG1 and IgG3 subclass and lack IgG4. Ulcerative colitis p-ANCA are myeloid specific. IIFT data indicate that the related antigen(s) seem(s) to be located not in the cytosol, but in the granules (most likely the azurophil granules) of the neutrophil. CONCLUSIONS--p-ANCA in ulcerative colitis have a different immunoglobulin subclass distribution than the ANCA of systemic necrotising vasculitis and necrotising and crescentic glomerulonephritis. This may point to differences in immune regulation between these diseases. Both cathepsin G and lactoferrin are recognised by a subpopulation of ulcerative colitis p-ANCA. In our series, eight out of 36 (22%) of ulcerative colitis associated p-ANCA react with lactoferrin and seven (19.5%) other sera with cathepsin G. None of them recognised both antigens. The main target antigen(s) of ulcerative colitis p-ANCA still remain(s) to be identified.     

Serum antibodies to Escherichia coli in subjects with ulcerative colitis.

It has been proposed that in ulcerative colitis the intestinal flora stimulates autoimmune reactions to colonic epithelium through shared specificities exposed in a `common antigen'' found in most Enterobacteriaceae. The present experiments aimed to resolve conflicting data as to whether patients with ulcerative colitis have selectively increased serum antibody titres to enterobacterial common antigen or E. coli 014, which is rich in enterobacterial common antigen. Antibody titres to enterobacterial common antigen and lipopolysaccharides of E. coli 014 and of five serotypes of E. coli which occur frequently in human faeces were measured by passive haemagglutination. Sera were obtained from patients with ulcerative colitis, age- and sex-matched controls and subjects with other gastrointestinal disorders. Serum titres to enterobacterial common antigen and E. coli 014 lipopolysaccharide were not increased significantly in subjects with ulcerative colitis but significant increases were observed in subjects with chronic liver disease without colitis. Patients with active ulcerative colitis, patients with chronic liver disease and subjects convalescent from Salmonella or Shigella infections all had significantly increased serum titres to the antigens as a group. Class-specific enhancement of passive haemagglutination indicated that the class distribution of serum antibodies was similar in subjects with ulcerative colitis and controls.     

Malakoplakia in ulcerative colitis.

Classic malakoplakia was found in the colon of a patient with a 30-year history of proven ulcerative colitis. She had undergone total proctocolectomy after failure of medical treatment to control her illness. Immunoperoxidase studies showed immunoglobulins and muramidase within the malakoplakic histiocytes, and electron microscopy showed bacteria resembling Escherichia coli in the same cells. Immunologic studies on the patient showed an unusually high E coli antibody titer (1:512) in her serum and reduced numbers of circulating T-lymphocytes with reduced cytotoxic activity. This case shows the paradoxical rarity of malakoplakia in ulcerative colitis and reaffirms the presence of an immunologic defect that may be pathogenetically significant.     

Immunological studies with different classes of mutants affected at the adenosine kinase locus in CHO cells

Adenosine kinase (AK) from CHO cells has been purified to homogeneity and specific antibodies to it have been raised in rabbits. Using this antibody, the presence of a specific cross-reacting protein (CRP) in cell extracts of different classes of mutants resistant to purine nucleoside analogs which are affected in AK has been investigated by the immunoblotting technique. Results of our studies show that 31 of the 32 independently selected class A AK^– mutants (obtained at high frequency in presence of adenosine analogs toyocamycin, tubercidin, 6-methylmercaptopurine riboside, or pyrazofurin and containing no measurable activity of AK in cell extracts) contained similar amounts of a specific CRP as seen in the parental AK⁺ cells. The CRP in the parental and different mutant cell lines has the same relative molecular mass as purified AK. Similar results were obtained with two mutants each of the class B and C type (selected in presence of C-nucleosides formycin A and formycin B), which are also affected in AK but show novel properties. The presence of equivalent amounts of the CRP in the vast majority of the class A mutants strongly indicates that the high frequency of those mutants in CHO cells is not a result of an epigenetic or deletion type of event, but that such mutants may contain missense types of mutations at a presumed mutational hot spot within the structural gene for adenosine kinase.     

Circulating antibodies to heat-shock protein 60 in Crohn''s disease and ulcerative colitis.

Heat-shock proteins (HSPs) are highly conserved immunogenic intracellular molecules that are induced by inflammatory mediators and may induce autoimmune phenomena in vivo. We have recently demonstrated the increased expression of HSP-60 in the colonocytes of patients with ulcerative colitis. To study further the role of HSP-60 in inflammatory bowel disease, we have now measured antibodies to recombinant mycobacterial HSP-65 (a member of the HSP-60 family) in patients with Crohn's disease, ulcerative colitis, healthy volunteers and, as disease controls, patients with confirmed bacterial diarrhoea. In comparison with healthy controls (n = 20; median level of 89 ELISA units; range 24-292), serum IgA HSP-60 antibodies were elevated in Crohn's disease (n = 21; 157; 57-364; P < 0.05) and active ulcerative colitis (n = 16; 188; 58-373; P < 0.01) but not bacterial diarrhoea (n = 10; 106; 51-285). Increased IgA HSP-60 antibody levels in patients with inflammatory bowel disease may occur as the result of HSP release from injured gut epithelium; alternatively, increased intestinal permeability could facilitate mucosal access of luminal antigens and the generation of cross-reactive anti-bacterial HSP antibodies.     

The metabolism of different immunoglobulin classes in irradiated mice

The catabolism of the different classes of immunoglobulins (Ig) of normal and irradiated mice was studied. A lethal dose of X-rays brought about no significant alterations in the catabolic rates of IgM and IgA, which kept to a time of about 12 hours. Contrariwise, the IgG (IgG₁, IgG_2a and IgG_2b) displayed a distinctly accelerated catabolism in the irradiated mice, the half-life of these Ig''s being reduced by approximately one-half. We propose an interpretation of this phenomenon, based, on one hand, on the great radiosensitivity of the intestinal epithelium and, on the other hand, on the mode of catabolism of IgM and IgA which appears to be different from that of IgG.     

Serum and salivary immunoglobulin A and free secretory component in ulcerative colitis

Patients with ulcerative colitis have been investigated for evidence of defects in secretory immunity. Total serum IgA concentration, serum IgA antibody titres to Candida albicans, salivary IgA and free secretory component concentrations have been measured in thirty-six patients with ulcerative colitis and thirty-six normal controls. None of the parameters was significantly different between patients with proctitis and patients with extensive colitis or between the colitis patients and normal controls.     

Circulating antibodies to the surface antigens on colon epithelial cells in ulcerative colitis.

The fluorescence activated cell sorter (FACS) was used for detecting circulating antibodies to the surface antigens on isolated colon epithelial cells (anti-colon antibodies) by indirect immunofluorescence. Anti-colon antibodies were found in the serum of 30 of 41 (73%) patients with ulcerative colitis. This incidence is much higher than one established in earlier reports by application of indirect immunofluorescence to colon tissue using the fluorescence microscope. The results suggest that FACS analysis is very useful for detecting antibodies to colon specific antigen.     

Appendiceal involvement in ulcerative colitis.

Little information has been published regarding appendiceal changes in ulcerative colitis of any extent. An early study has shown the appendix to be involved in over 50% of all cases of ulcerative colitis for which a proctocolectomy was performed, but the extent of the colitis was not always defined. While some investigators have found appendiceal involvement only in continuity with adjacent involved cecum, others believed it may occur as a skip lesion. In this study, the colons and nonobliterated appendices of 87 patients who underwent total proctocolectomy for ulcerative pancolitis were examined to determine the frequency of appendiceal involvement and to determine the frequency with which such involvement truly occurs as a skip lesion. All 87 cases had ulcerative pancolitis, and 66 (62%) had colitic changes in the appendix. In the remaining 21 cases, there was no appendiceal inflammation. In all cases in which the appendix was involved, the cecum was also involved. Cecal activity or lack of activity correlated with appendiceal activity in 52 of the 87 cases (60%). Of the 35 cases in which there was some discrepancy in disease activity between the appendix and cecum, nine had more active disease in the appendix, and 26 had greater activity in the cecum, but in none of the cases where the cecum was normal or near normal was the appendix more severely involved. These data suggest that appendiceal involvement in resected ulcerative pancolitis always occurs in continuity with adjacent involved cecum, although there may be differences in disease activity between the two sites.     

Immunological studies of an atypical (myeloma) immunoglobulin

An 8S myeloma component, isolated from serum of a patient with myelomatosis is described, which appears to have no antigenic determinants in common with human, α-, δ-, γ- or μ-polypeptide chains as revealed by immuno-electrophoresis and Ouchterlony gel diffusion analysis.

The myeloma protein migrates in the fast γ-region on electrophoresis at pH 8.6 and has an elution volume on Sephadex G-200 similar to that of 6.5S IgA.

The isolated myeloma component has an approximate molecular weight of 200,000 and a total carbohydrate content of 10.7 per cent.

Reduction with β-mercaptoethanol and acid dissociation yields light polypeptide chains of Type L and a carbohydrate-rich component, in the ratio of 1:4.

Antisera specific to determinants on the heavy chains of the myeloma protein showed no reaction with the immunoglobulins A, D, G or M. Instead unique determinants were found on the heavy polypeptide chains.

     

Immunological studies on Kawasaki disease. I. Appearance of Hanganutziu-Deicher antibodies

Sera of patients with Kawasaki disease were studied for heterophile antibodies by means of enzyme immunoassay (EIA) with enzyme conjugated antisera to human IgM, IgG, IgA and IgE. Antibodies of IgM (43%), IgG (3%), IgA (11%) and IgE (49%) classes were demonstrated that combined with high molecular weight glycoprotein (HMWGP) of bovine red blood cells (BRBC) one of the antigenic preparations of the Hanganutziu-Deicher (H-D) heterophile system. Studies on sequential sera of the patients revealed that HMWGP antibodies of IgM and IgE classes began to appear in the second week, reached their peaks in the third week of the disease and declined gradually thereafter. Absorption studies on the positive sera showed that the HMWGP antibody activities were abolished by BRBC, sheep red blood cells and guinea-pig kidney tissues, confirming H-D specificity of these antibodies. EIA inhibition studies showed that the antibody activity was inhibited by HMWGP and partially by asialo-HMWGP and NGNA ganglioside rich preparation of BRBC but not by purified Paul-Bunnell or Forssman antigens. These results indicate that the H-D antibodies under investigation consist of antibodies of two different specificities; one directed against asialo-HMWGP and the other NGNA ganglioside of BRBC. Circulating immune complexes (IC) were demonstrated in 23% of the patients by means of anti-antibody inhibition test. Evidence was presented that IC in the sera of five patients were composed of H-D (HMWGP) antigen and its corresponding antibodies.