Correlation between Serological and Mucosal Inflammatory

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1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1994, p Vol. 1, No X/94/$ Copyright C) 1994, American Society for Microbiology Correlation between Serological and Mucosal Inflammatory Responses to Helicobacter pylori GUILLERMO I. PEREZ-PEREZ,l* WILLIAM R. BROWN,2 TIMOTHY L. COVER,1'3 BRUCE E. DUNN,4 PING CAO,1 AND MARTIN J. BLASER1' 3 Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee ; Division of Gastroenterology, Department of Veterans Affairs Medical Center, and University of Colorado School of Medicine, Denver, Colorado ; Infectious Disease Section, Department of Veterans Affairs Medical Center, Nashville, Tennessee ; and Laboratory Service, Veterans Administration Medical Center, Milwaukee, Wisconsin Received 6 December 1993/Returned for modification 1 February 1994/Accepted 23 February 1994 In 82 patients who underwent gastroduodenoscopy, acute and chronic gastric mucosal inflammation was scored for severity, and systemic humoral immune responses to Helicobacter pylori antigens were assessed by enzyme-linked immunosorbent assays. On the basis of culture, gastric histology, and serologic evaluation, 33 patients were classified as H. pylori infected and 36 were classified as uninfected. Thirteen patients had negative cultures and stains but were seropositive and were analyzed separately from the other two groups. Specific serum immunoglobulin G (IgG) subclass responses to H. pylori whole-cell antigens and specific IgG responses to the 54-kDa heat shock protein homolog (Hp54K) and vacuolating cytotoxin were significantly greater in infected than in uninfected patients as were specific IgA responses to whole-cell antigens and cytotoxin (P < 0.001). Among the H. pylori-infected persons, serum IgG responses to Hp54K and to the vacuolating cytotoxin were correlated with acute mucosal inflammatory scores. In contrast, serum IgA responses to whole-cell sonicate and to vacuolating cytotoxin were inversely related to chronic inflammatory scores. By multivariant regression analysis, only specific serum IgG responses to Hp54K correlated with severity of inflammation (both acute and chronic; P < 0.001); these responses may be markers of inflammation or these antibodies could play a direct role in the pathogenesis of H. pylon-induced inflammation. Helicobacter pylon infection is associated with chronic gastritis and gastroduodenal ulceration and with adenocarcinoma of the distal stomach (1, 15, 25), but the determinants for particular outcomes of infection have not been characterized. Sipponen (28) has suggested that gender, nonsecretor status, and antral gastritis are independent host risk factors for peptic ulcer; however, H. pylon virulence factors also could be important (2). Soluble cellular antigens such as urease and heat shock protein (10, 11), a vacuolating cytotoxin (4, 20), and, more recently, a 128-kDa protein (CagA) associated with cytotoxin production (32) have been suggested as possible inducers of an inflammatory reaction in the gastric mucosa (21, 22) and could explain how bacteria living in the mucus layer can produce histologic lesions in the full thickness of the mucosa (2, 22). In addition, it has been suggested that intensity and specificity of the mucosal immune response may correlate with the level of tissue inflammation (19). In this study, we examined a possible relationship between the type and severity of gastric inflammation and the systemic humoral immune response to particular H. pylon antigens. MATERIALS AND METHODS Patients studied. Eighty-two patients presenting to the Gastroenterology Service of the Denver Department of Veterans Affairs Medical Center (n = 65) and University of Colorado Hospitals (n = 17) between January and December 1987 underwent endoscopy for diagnosis of upper gastrointes- * Corresponding author. Mailing address: Vanderbilt University School of Medicine, Division of Infectious Diseases, A-3310 Medical Center North, Nashville, TN Phone: (615) Fax: (615) tinal complaints. Eighty-one patients had not received antimicrobial therapy during the month prior to evaluation; one patient who received antimicrobial therapy was not infected with H. pylon. Of the total number of patients, 38% had been treated with H2 blockers. The H. pylon-infected patients (54.5%) had been treated with H2 blockers significantly more often than had the uninfected patients (27.8%; P = 0.009). Tissue samples obtained from two biopsies of the gastric antrum were processed for culture of H. pylon and for histologic study. Serum samples were frozen at -20 C. Culture of H. pylori. Gastric specimens for culture were placed in a single vial of 20% sterile glucose and then smeared directly onto H. pylon-selective media (containing 7% horse blood, 1.8% IsoVitaleX [BBL], 10,ug of vancomycin per ml, 10,ug of nalidixic acid per ml, and 2,ug of amphotericin B per ml) and onto nonselective chocolate agar plates. The plates were incubated under microaerobic conditions at 37 C for up to 10 days. The isolates were identified as H. pylon by Gram stain morphology and by urease, oxidase, and catalase positivity (23). Histology. Biopsy specimens were placed into 10% formalin and embedded in paraffin. Hematoxylin-and-eosin-stained sections were graded blindly for the presence of mononuclear cells (chronic inflammation) and polymorphonuclear leukocytes (acute inflammation) as described by Hazell et al. (16). All biopsy slides were read blinded by a single pathologist (B.E.D.). For chronic inflammation, the grades were defined as follows: 0, no mononuclear cell excess; 1, only occasional mononuclear cells present in a patchy distribution; 2, mononuclear cell infiltration intermediate between grades 1 and 3; 3, very dense infiltration of mononuclear cells throughout the entire section. Parallel criteria for polymorphonuclear leukocytes were used for grading acute inflammation. Additional 325

2 326 PEREZ-PEREZ ET AL. sections were processed to detect H. pylon by using a modified Gram stain in which 0.5% carbol fuchsin was used as a counterstain. Serological assay for H. pylori antigens. Serum antibodies to sonicated H. pylon whole-cell antigens (10 p.g/ml) were detected by using an enzyme-linked immunosorbent assay (ELISA) and screening serum dilutions of 1:800 and 1:100 for immunoglobulin G (IgG) and IgA, respectively, as described previously (27). For determination of the IgG and IgA subclass responses, sera were tested at 1:100 dilutions, and peroxidase-conjugated sheep anti-human IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2 (The Binding Site, San Diego, Calif.) were used as the second antibodies. Human myeloma IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2 proteins were used to develop a standard curve (Calbiochem, San Diego, Calif.), and results for patient specimens were expressed as concentration values (micrograms per milliliter) for each immunoglobulin subclass. Human immune response to the vacuolating cytotoxin and cpn6o heat shock protein of H. pyloni. H. pylon (7, 20) was used for the purification of the vacuolating cytotoxin as described previously (4). In brief, the cytotoxin was purified from culture supernatant by ammonium sulfate precipitation, followed by hydrophobic interactive chromatography, gel filtration with a Superose 6 column (Pharmacia), and ionexchange chromatography (4). The 54-kDa cpn6o heat shock protein homolog (Hp54K) was purified from water-extractable proteins of H. pylon by size exclusion and anionexchange chromatographies as reported previously (10, 11). Immulon 2 plates (Dynatech Laboratories, Inc., Alexandria, Va.) were coated with 30 ng of HpS4K per well (11) or 15 ng of cytotoxin per well (4, 6), and human sera diluted 1:100 were examined by ELISA as described previously (27). Peroxidaseconjugated goat anti-human IgG and IgA (Tago, Inc., Burlingame, Calif.) were used as the second antibodies. Statistical methods. Distributions of optical density ratios were compared by Student's t test (two tailed) for independent variables. Single variables were studied by one-way analysis of variance, and multiple variables were analyzed by multiple linear regression. Correlation between variables was determined by statistical data analysis SPSS/PC+4.0 (SPSS, Inc., Chicago, Ill.). Differences in proportions were analyzed by chi square or Fisher's exact test. RESULTS Characteristics of the study population. Patients were defined as H. pylon infected if H. pylon was cultured, if characteristic organisms were visualized in tissue specimens, or if serum IgG values exceeded a previously defined and validated (8, 9, 29) threshold. On the basis of these criteria, 46 patients (56.1%) were found to be H. pylon infected, and 36 (43.9%) were not. Thirteen of the 46 patients were found to be infected on the basis of serology alone and were therefore analyzed separately from the other two groups. Culture, histology, and serology detected infection in 60.6, 93.9, and 93.9%, respectively, of the 33 unequivocally infected patients; all 33 patients were positive in at least two assays. The 33 infected and 36 uninfected persons were similar in age (54.4 ± 2.1 versus 53.9 ± 3.0 years, respectively). Peptic ulceration was present in five of the H. pylon-infected persons and in three of the uninfected persons. Two of the H. pylon-infected persons had duodenal ulceration, and three had gastric ulceration at the time of endoscopy. Of the uninfected persons, two had duodenal ulceration, and one had gastric ulceration. Of the 33 H. pylon-infected patients, 32 (97.0%) showed gastric inflammation on histological examination. In contrast, TABLE 1. Association between the H. pylon status and presence of inflammation in 82 patients Patients with Presence of H. pylon by: No. of gastric patients inflammation in group Culture Stain Serology No. % + + +a a Sixteen patients were positive, and two were negative. CLIN. DIAGN. LAB. IMMUNOL. only 4 of the 36 uninfected patients showed evidence of gastric inflammation (P < ) (Table 1); one of these patients had duodenal ulceration. Not surprisingly, the H. pylon-infected patients had significantly higher scores for both acute ( versus 0.05 ± 0.04) and chronic (1.21 ± 0.13 versus 0.11 ± 0.05) inflammation than did the uninfected persons (P < for each comparison). For the 33 infected persons, antral biopsies from 22 (66.7%) showed evidence of acute inflammation, 28 (84.8%) showed evidence of chronic inflammation, and 17 showed both types. Acute inflammation scores of 0, 1, 2, and 3 were present in 33, 52, 15, and 0%, respectively, of the H. pylon-infected patients. Chronic inflammation scores of 0, 1, 2, and 3 were present in 15, 55, 24, and 6%, respectively, of the H. pylon-infected patients. Nine (60%) of the 15 H. pyloriinfected patients who had either chronic or acute inflammation scores of zero had been taking H2 blockers. The 13 patients in whom H. pyloni was diagnosed solely by serological criteria showed gastric inflammation more frequently (54%) than did patients who were negative for H. pyloni (4 [11%] of 36) by all three criteria used (P = 0.005). Serum antibody response to H. pylori antigens. Compared with the 36 uninfected persons, the 33 unequivocally H. pylon-infected persons had significantly higher responses to H. pylon whole-cell antigens in total IgG, IgGl, IgG2, IgG3, IgG4, total IgA, and IgAl classes (Table 2). Among H. pylorz-infected persons, IgG subclass responses to the whole-cell antigens were not significantly different for those with peptic ulcer disease and those with gastritis alone (data not shown). Moreover, no significant differences were observed in the antibody response to H. pylon-specific antigens between infected patients with peptic ulcer disease (n = 5) and those patients with gastritis alone (n = 28). However, all infected patients with peptic ulcer disease possessed antibody against the cytotoxin compared with 10 of the 28 patients with gastritis alone (P = 0.012, Fisher's exact test). IgG but not IgA responses to Hp54K were significantly (P < 0.001) greater in H. pyloni-infected persons than in uninfected persons. H. pyloni-infected persons had significantly higher levels of both IgA and IgG to the vacuolating cytotoxin than did uninfected persons (P < 0.001). There were significantly higher responses to H. pyloni whole-cell antigens in total IgG, IgGl, IgG2, IgG3, IgG4, and total IgA for the 13 patients in whom H. pyloni was diagnosed solely by serological criteria than in the 36 uninfected persons. Similar to the responses of the infected group, the IgG response to HpS4K and the IgA and IgG responses to the vacuolating cytotoxin were significantly greater (P < 0.001) in these 13 patients than in uninfected persons. Relation of specific serologic markers to inflammation in H. pylori-infected persons. We next focused exclusively on the 33 unequivocally H. pylon-infected persons to determine whether

3 VOL. 1, 1994 GASTRIC INFLAMMATION AND H. PYLORI IMMUNE RESPONSE 327 TABLE 2. Comparison of the serum antibody responses to H. pylon antigens in 69 endoscoped patients Antibody Antigen H. pylon infected (n = 33) H. pylon uninfected (n = 36) (sub)class OD Concn (,ug/ml) OD Concn (,ug/ml) IgG Whole-cell sonicate 2.34 ± ± 0.04 <0.001 IgGl Whole-cell sonicate 0.10 ± ± <0.001 IgG2 Whole-cell sonicate 0.06 ± ± <0.001 IgG3 Whole-cell sonicate 0.07 ± ± <0.001 IgG4 Whole-cell sonicate ± ± <0.001 IgA Whole-cell sonicate ± 0.12 <0.001 IgAla Whole-cell sonicate 0.05 ± ± Ig2a Whole-cell sonicate 0.08 ± ± 0.01 NSb IgG Hp54K 0.43 ± ± 0.01 <0.001 IgAc Hp54K 0.28 ± ± 0.03 NSb IgG Cytotoxin 0.59 ± ± 0.02 <0.001 IgA Cytotoxin 0.62 ± ± 0.02 <0.001 a Five H. pylon-infected and five H. pylori-uninfected persons were not tested. b NS, not significant (P > 0.05). c One of the H. pylon-infected persons was not tested. the serologic assays could further discriminate in relation to type and severity of their gastric inflammation. Analysis of IgG or IgA subclass responses to H. pylon whole-cell antigens failed to demonstrate any association with gastric inflammation (data not shown). By univariant analysis, the magnitude of the IgG response to Hp54K was higher in those patients with the highest (grade 2) acute inflammation scores than patients with lower (grade 1) scores (0.63 ± 0.15 versus 0.36 ± 0.05, P = 0.02). Similarly, patients with chronic inflammation (grades 1 to 3) had higher IgG responses to Hp54K than infected patients without chronic inflammation (i.e., grade 0; versus 0.26 ± 0.09, P = 0.057). Patients with the highest acute inflammation scores (grade 2) had higher IgG responses to the cytotoxin than did patients with lower scores (grades 0 and 1; 0.81 ± 16 versus 0.55 ± 0.07, P = 0.09). There was no relationship between the magnitude of IgA response to the cytotoxin and acute inflammation scores, and IgA responses to HpS4K were not correlated with either acute or chronic inflammation scores. However, patients with lower chronic inflammation scores (grades 0 and 1) had higher IgA responses to sonicated whole-cell antigens than patients with higher chronic inflammation scores (grades 2 and 3; versus 1.55 ± 0.19, P = 0.02). Patients without chronic inflammation (grade 0) also had higher IgA responses to cytotoxin than did patients who had chronic inflammation present (grades 1 to 3; 0.86 ± 0.18 versus 0.57 ± 0.06, P = 0.1). To further evaluate the correlations observed in the univariant analyses, multiple linear regression analysis was performed with the 69 patients with defined H. pylon status (Table 3). In this analysis, the magnitude of the serum IgG response to Hp54K was positively correlated with both acute and chronic inflammation scores (r = 0.61 and 0.61, respectively; P TABLE 3. Multiple linear regression analysis of serum antibody responses in relation to gastric inflammation score in 69 H. pyloninfected patients Acute Chronic inflammation Antigen Antibody inflammation ~class r P r P Hp54K IgA IgG < Cytotoxin IgA IgG < 0.001); however, none of the other humoral responses correlated with inflammation scores in these multivariant analyses. In the evaluation of the 13 patients who were only seropositive for H. pylori antigens and not tissue positive, we compared the immune responses to H. pylori antigens of the seven patients with gastric inflammation with those of the six patients without gastric inflammation. The only difference observed was the IgG response to HpS4K antigen, which was slightly but not significantly higher in patients with gastric inflammation than in patients without inflammation (0.46 ± 0.07 versus 0.30 ± 0.06, P = 0.07). DISCUSSION The hypothesis we wished to explore in this study was that the humoral and tissue responses to H. pyloni infection were correlated. Since eradication of H. pylori leads to concomitant reductions in tissue inflammation (33) and decreases the levels of serum antibodies (18), the hypothesis that these phenomena are linked in untreated persons is plausible. Because serologic responses to particular antigens or particular immune subtype responses might correlate better with tissue responses than others, we explored both possibilities. One limitation of this study was that only two biopsy samples were obtained from each patient, which hinders the correct classification of infection because of the patchy nature of gastritis. However, serological evaluation, in essence, involves sampling the entire stomach (30) and can correct for this phenomenon. Another limitation of this study is that only 60.6% of the infected patients were culture positive despite the demonstration of organisms by histological stain in 93.9% of infected patients. Moreover, the same 93.9% of the infected patients were diagnosed as being infected on the basis of an IgG serologic test with a high degree of accuracy (-93%) (8, 9, 29, 30). The false-negative culture results in many patients are attributable to the suboptimal culture techniques which were in use at the time that endoscopies were performed. Nevertheless, because of uncertainties about the persons who were only seropositive, we excluded them from subsequent analyses. Not surprisingly, IgG and IgA subclass responses paralleled total IgG and IgA responses in their ability to distinguish between H. pylon-infected and uninfected persons, but they did not offer any advantage in the differentiation of patients with different degrees of inflammation. Bontkes et al. have reported recently that patients with duodenal ulcers have higher levels

4 328 PEREZ-PEREZ ET AL. of H. pylon-specific IgG2 than do other infected patients (3), but perhaps because of small patient numbers, we did not find a significant difference in this study. Although the IgG ELISA using whole-cell antigens is a highly accurate diagnostic test (8, 9, 27, 30), the magnitude of optical density values did not correlate with the severity of tissue inflammation. Systemic IgA responses to H. pylon antigens are not as useful for the diagnosis of H. pylon infection as is the systemic IgG response (14), and the diversity observed is consistent with independent regulation of the systemic and local humoral responses. Specific IgA responses could potentially block immunopathogenic IgG responses; the negative correlation we observed between the specific IgA response to multiple H. pyloni antigens (wholecell and cytotoxin preparations) and the severity of chronic inflammation, although not statistically significant, is consistent with this hypothesis. The lack of association that we observed between IgG antibody levels to whole-cell antigens and inflammation scores could reflect the difficulty of recognizing a true relationship with a single antigen because of high background reactivity to other H. pylori antigens. For that reason, we examined two purified proteins that could potentially play a role in gastric inflammation. Antibodies to the cytotoxin were present more often in the sera of infected than uninfected persons, confirming in vivo cytotoxin production (6). Although not statistically significant, there was a trend toward correlation between acute inflammation and IgG serologic responses to the cytotoxin. Two recent studies from our laboratory have also demonstrated an association between cytotoxin production and acute inflammation (5, 26). Moreover, a correlation between infection with a cytotoxin-producing strain and a serologic response to the cytotoxin also has been demonstrated (5). The cpn6o class of heat shock proteins (chaperonins) is highly conserved in life forms ranging from bacteria to mammals (12). Despite their ubiquity and functional and structural conservation, cpn60 molecules of other bacteria are highly antigenic (34). Furthermore, these molecules have been implicated as immunogens involved in the immunopathogenesis of chlamydial (31) and mycobacterial (17) infections. Thus, it is reasonable to postulate that the H. pylon cpn60 homolog, Hp54K (11), could have similar properties. In this study, there was a significant association of the magnitude of the IgG response to Hp54K and both acute and chronic mucosal inflammatory scores. These data may indicate that there is a biological relationship between the magnitude of humoral responses and the magnitude of cellular responses to Hp54K. Alternatively, antibody to Hp54K may play a direct pathogenic role in the process of gastric inflammation. 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