JOURNAL OF PATHOLOGY, VOL. 159: 231-237 (1989)

CELLULAR AND HUMORAL IMMUNE RESPONSES IN CAMPYLOBACTER

P YLORI-ASSOCIATED CHRONIC GASTRITIS JAVED IQBAL KAZI*, RAJA S I N N I A H ~ , N. A. JAFFREY*, s. M. ALAM*, v. Z A M A N ~ , s. J. Z U B E R I ~ AND

A. M. K A Z I ~

*Department of Pathology, Jinnah Postgraduate Medical Centre, Karachi, Pakistan; ?Department of Pathology, National University of Singapore; $Pakistan Medical and Research Council; $Department of Medicine,

Jinnah Postgraduate Medical Centre, Karachi, Pakistan

Received 5 December 1988 Accepted24 May 1989

SUMMARY

Gastric cellular and humoral immune responses investigated by immunoperoxidase staining of 53 antral biopsies showed significant differences in Campylobacter pylori-associated gastritis as compared with non-bacterial chronic gastritis and normal controls. IgA, secretory component, and complement C3 coated bacilli were seen in all cases of active chronic gastritis. C3 was always associated with coating by IgA, IgM, or both, which were rarely seen in gastritis without polymorphonuclear neutrophil infiltration. Intraepithelial mononuclear cellular infiltration was seen in 18 of 26 cases of C. pylori-associated chronic gastritis. The intraepithelial mononuclear cells stained positively for T cells and histiocytes.

KEY WORDS-Campylobacter pylori-associated gastritis, cellular immunity, humoral immunity, immunoperoxidase.

INTRODUCTION

The presence of ‘spiral bacteria’ on gastric mucosa has been noted by histopathologists for many years.’-2 Interest in these bacteria gained fresh impetus following their recognition as campylo- bacter species using microbiological technique^.^ The organism was first named Campylobacter pylori4 C. pylori infection has been associated with gastric ulcer, duodenal ulcer, non-ulcer dyspepsia, and gastritis.’-’ Reports from many parts of the world have been remarkably consistent and the evi- dence for C. pylori as a cause of gastritis is accumu- lating rapidly. Marshall et ingested a culture of C. pylori and developed symptomatic gastritis associated with mucosal colonization, thereby ful- filling Koch’s third and fourth postulates. Systemic and local immune responses to the infection have been described,’-’’ but further investigation of

Addressee for correspondence: Professor Raja Sinniah, Department of Pathology, National University Hospital, National University of Singapore, Lower Kent Ridge Road, Singapore 05 1 I .

0022-3417/89/11023147 $05.00 0 1989 by John Wiley & Sons, Ltd.

immune responses at the mucosal surface is likely to help elucidate the role of C. pylori in chronic gastri- tis. Our aim in this study was to investigate the possible humoral and cellular immunity in C. pylori- associated chronic gastritis, and to compare it with histologically normal mucosa and with cases of chronic gastritis where no C. pylori were detected.

MATERIALS AND METHODS

Fifty-three antral biopsies, including full thick- ness of mucosa, were studied by the immunoperoxi- dase staining method. The mucosal distribution, intensity of staining, and coating of the organisms by IgA, IgG, IgM, secretory component, comp- lement (C3) and lysozyme were noted. The cells in the lamina propria and the intensity of mucosal staining were graded on an arbitrary scale of0 to 3 + .

The 53 antral biopsies included 46 cases of chronic gastritis (26 cases with and 20 without C. pylori colonization), and 7 samples of histologically normal antral mucosa which served as controls.

232 J. I. KAZI ETAL.

Histopathology Formalin-fixed, paraffin-embedded 4 pm sections

were stained with haematoxylin and eosin (H&E) for the presence and grading of chronic gastritis.16 The biopsies were diagnosed as normal when inflammatory cells were rarely seen, or when few lymphoid cells were present but within normal limits and with no other evidence of inflammation. Chronic gastritis showed an increased number of lymphoid cells, or other signs of cell damage, and active chronic gastritis showed an obvious increase in polymorphonuclear neutrophils in the mucosa or few polymorphonuclear cells seen invading the epithelium. The epithelial invasion by polymorpho- nuclear and mononuclear cells was also recorded separately. ’’

Sections stained with a modified Giemsa stain were examined for small curved bacilli on the mucosal surface and in the superficial and deep pits. The number of bacilli was graded from 0 to 3 +: grade 0 with no characteristic bacilli; grade I + with occasional curved bacilli found after a thorough search; grade 2 + with scattered bacilli in most high power fields or occasional groups of them; and grade 3 + with numerous bacilli in most high power fields.

Immunoperoxidase staining Biopsy specimens were stained by the three-stage

peroxidase-antiperoxidase (PAP) method with rabbit polyclonal antisera to human IgA, IgM, IgG, secretory component, and A 1 AT (for histiocytes) (DAKO). Mouse monoclonal antibodies MB2 (Clonab) and UCHL-1 (DAKO) were used for B cells and T cells, respectively, which were stained by the indirect immunoperoxidase method. We used AlAT, a known marker for histiocytes.I8 The immunoreactivity of monoclonal antibody MB2 was stronger than that of monoclonal antibody MBl for identification of B cells,” and murine monoclonal antibody UCHL-1 was useful in identig fying T cells” in formalin-fixed, paraffin-embedded tissues. UCHL-1 identifies an antigen present on most thymocytes. A subpopulation of resting T cells within both the CD4 and CD8 subsets and on mature activated T cells.2’ Sections were trypsinized in 0.1% CaCl,, pH 7.8, for 20 min at 37°C for all antibodies except MB2 and UCHL-1. All primary antibodies except MB2 and UCHL- 1 were incubated first with swine anti-rabbit antibodies followed by rabbit-PAP complex. MB2 and UCHL-1 were incubated first with rabbit anti-mouse horseradish

peroxidase conjugate followed by swine anti-rabbit horseradish peroxidase conjugate. Sections with all primary antibodies were incubated overnight at 4°C. All incubations were carried out in a humid chamber. Negative controls consisted of sections of normal non-colonized mucosa, and C. pylori col- onized mucosa with omission of primary antibody.

Immunoperoxidase staining results were evalu- ated without knowledge of histological gradings. Immunoperoxidase staining of bacilli for IgA, IgM, IgG, secretory component, complement C3, and lysozyme were recorded and the intensity of staining was graded for bacilli on surface, superficial and deep pits, separately. The mucosal distribution of all the antigens was noted and the intensity of staining was graded in different zones which included (1) superficial epithelium, (2) foveolar epithelium, (3) isthmus, (4) glandular epithelium, and (5) lamina propria. The staining intensity was graded on an arbitrary scale as grade 0 to 3 + .” Grade 0 indicated no or negligible specific staining; grade I + oc- casional and variable staining; grade 2+ more regular and intense staining; and grade 3 + intense staining with considerable extension throughout the evaluated zone taking the whole section into account. The grades of each zone were added to quantify the staining intensity giving a possible range of 0 to 15 for each marker.

Statistical methods

and the Kruskal Wallis test. Statistical analyses were done using the X2-test

RESULTS Table I shows significant differences in cellular

and humoral immune responses except for histio- cytes in C. pylori-associated gastritis as compared with normal stomach and non-bacterial gastritis. The humoral responses of IgA and secretory com- ponent, and the cellular responses of T cells and polymorphonuclear cells were particularly high.

The staining of bacilli for all immunoglobulins was more intense on the surface of the gastric mucosa (Fig. 1). The intensity of staining and the number of C. pylori as seen by PAP staining de- creased gradually in the superficial and deep pits. In most of the cases, the bacilli in the deep pits were not stained except in a few cases of active chronic gastri- tis where C. pylori in the deep pits were weakly stained for secretory component and lysozyme. Unstained bacteria were always seen beside the stained bacteria.

C. PYLORI-ASSOCIATED CHRONIC GASTRITIS 233

Table I-Humoral and cellular immune responses in C. pylori-associated gastritis, compared with controls and non-bacterial gastritis (Kruskal Wallis test)

Markers Calculated x2 (2df)

H-value 0.05 P value

IgA IgM IgG Secretory component Lysozyme Histiocytes T cells B cells Polymorphonuclear cells

37.3887 28.3098 25.2336 28. I345 24.1741 2.7796

11.3781 7.2852 9.4999

5.991 5.991 5.91 1 5.991 5.991 5.99 1 5.991 5.99 1 5.991

< 0.005 < 0.005 < 0.005 < 0.005 < 0.005

NS < 0.005 <0.01 <0.01

NS=Not significant.

Out of 26 cases infected with C. pylori, 16 had active chronic gastritis and 10 inactive chronic gastritis. gastritis; of 20 cases of non-bacterial gastritis, 4 had

active chronic gastritis and 16 had inactive chronic

Table I1 shows the distribution of staining of C. pylori by humoral factors in the different grades of gastritis. IgA, secretory component, and comp- lement were seen in all cases of active chronic gastri- tis. Complement C3 was always associated with IgG, IgM, or both, and was seen only in two cases of chronic gastritis without polymorphonuclear neutrophils. C. pylori stained for C3 were always surrounded by polymorphonuclear cells (Fig. 2) in cases of active chronic gastritis.

In the chronic gastritis group, immunoglobulins were seen in cells as well as in the intercellular regions of the lamina propria (Fig. 3). Patchy areas of epithelium were also stained for all immuno- globulins. Secretory component, consistently absent in normal mucosae, was seen in the epithelium in gastritis, with greater intensity in the isthmus zone. In chronic gastritis, the staining for lysozyme in pyloric glands became intense and the isthmus zones and histiocytes also took up the stain. The staining for lysozyme was more intense in C. pylori- associated chronic gastritis than in non-bacterial gastritis (Figs 4 and 5).

Table 111 shows a significant number of C. pylori- associated chronic gastritis cases with intraepithelial invasion by mononuclear cells, polymorphonuclear neutrophils, T cells, and histiocytes. The T cells, B cells, and histiocytes stained intensely, and only intracellular staining was noted. Figure 6 shows the intensity of humoral and cellular immune responses

with and without associated C. pylori infection.

Fig. 1-Immunoperoxidase stain showing bacilli coated by IgG (arrows). Some of the bacilli are unstained (immunoperoxidase stain)

in normal gastric biopsies and in chronic gastritis

234 J. I. KAZI ETAL.

Table 11-Bacterial staining by different humoral factors in C. pylori positive gastritis (n = 26)

Grades of gastritis

No. of Inactive chronic Active chronic positive gastritis gastritis

Markers cases (10) (16) P value

IgA 22 (85%) 6 IgG 17 (65%) 2 IgM 16(61%) 2

Lysozyme 15 (57%) 2 c3 18 (69%) 2

Secretory 22 (85%) 6 component

16 co.01 15 < 0.00 1 14 < 0.00 1 16 co.01

13 <0.01 16 < 0.001

Fig. 3-Immunoperoxidase staining of IgA in a case of C. pylori- associated gastritis showing staining in the mucosal epithelium and lamina propria, and in the cellular infiltrates. The total grade of staining intensity in this case was 6

Fig. 2-Immunoperoxidase stain showing numerous bacilli coated with C3 (arrows). Polymorphonuclear cells (arrow-heads) are seen among the organisms, many of which are unstained

DISCUSSION C3, and lysozyme were easily identified in the gastric mucosa. Immunoperoxidase labelling of C. pylori by anti IgA, IgM, and IgG has been reported.23 Unlabelled organisms were always present with the

C . pylori stained by anti-immunoglobulins IgA, IgG and IgM, secretory component, complement

C. PYLORI-ASSOCIATED CHRONIC GASTRITIS 235

Fig. 4-Immunoperoxidase staining for lysozyme in a case of chronic gastritis without C. pylori infection

Fig. 5-Immunoperoxidase staining for lysozyme in a case of chronic gastritis associated with C. pylori infection

labelled bacilli. In the presence of negative controls and unlabelled bacteria in each biopsy specimen, it seems unlikely that the positive staining of the bacilli is a non-specific reaction.

Type B chronic gastritisI6 is found predominantly in the antrum and is associated with hyperchlor- hydria and peptic ulceration. Thus far, this category has been considered idiopathic although irritants, both exogenous such as hot drinks and spices, and endogenous like bile reflux, are implicated as the cause of the inflammation.

C. pylori is a recently recognized organism which has been implicated as an infective cause of this type of gastritis. The gastric mucosal immunity, ineffec- tive against physical and chemical injuries, and expected to mount a heightened response against a bacterial pathogen, is in keeping with our findings that the gastric mucosal immune response was sig- nificantly higher in C.pylori-associated gastritis than in non-bacterial gastritis. Moreover in vivo adsorp- tion of host immunoglobulins, secretory compo- nent, lysozyme, and activation of complement C3 favour a causative role for C. pylori in type B

chronic gastritis. A distinctive form of chronic gas- tritis, ‘lymphocytic gastritis’, is characterized by marked infiltration of the surface and pit lining epi- thelium by mature T lymphocyte^.^^ c. pylori were detected in 41 per cent of cases of ‘lymphocytic gas- tritis’, but all except two of the 1 I cases tested had serological evidence of C . pylori infection. It was thus concluded that lymphocytic gastritis might also represent an abnormal response to a local anti- gen, possibly C.pylori, to which the patients become sensitized.

IgA is the main protective immunoglobulin on the gastric mucosa and inhibits bacterial adherence to epithelial cells.25 It is capable of mounting antibody-dependent cell mediated activity against enteropathogenic bacteria.26 Secretory IgA anti- bodies synergize with IgG in promoting antibody- dependent cell mediated cytotoxicity by human polymorphonuclear neutrophils, monocytes, and lymphocyte^.^' In our study, C. pylori-associated chronic gastritis showed the maximum immune re- sponse by IgA which was deposited on the bacterial surface in 22 of 26 cases.

236

7

J. I. KAZI ET AL.

0 0

6 * 0 I * 0 4 0 3 . * I . *

Table 111-Number of cases of chronic gastritis showing intraepithelial infiltration by cells in different groups

I 0

Type of intraepithelial cellular infiltrate

I 0

Gastritis Gastritis without C. pylori with C. pylori

(20) (26) P value

Mononuclear cells (H&E) Polymorphonuclear cells (H&E) T cells (immunoperoxidase) Histiocytes (immunoperoxidase)

18 <0.01 16 <0.01 15 < 0.05 6 < 0.05

0

Q 0

0

O *

* .-* 0 1 2 3 4 5 6 7 8 9 10 0 I 2 3 4 5 6 7 8 9 I0 11 I? 13 0 1 2 3 4 5 6 7 1 )

on 1-axie: m of cases m r u l group 00 Y-axis: Grade of Staining Gaetritis without C.Pylori

o Gastritis with C.Pylori

Fig. &Humoral and cellular immune responses in different groups of chronic gastritis and in controls

The association of C. pylori infection and the presence of polymorphonuclear neutrophils has been seen in chronic gastritis.28 Others have observed phagocytosed C. pylori in polymorpho- nuclear neutrophils on the mucosal ~urface. '~ The detection of IgG, IgM, and complement on bacterial surface was found to have different distributions in chronic gastritis with and without polymorpho- nuclear neutrophils. Complement was detected in all cases of active chronic gastritis, and was always associated with either IgG, IgM, or both. On the other hand, IgG, IgM, and complement were detected in only two cases of gastritis where poly- morphonuclear neutrophil infiltration was not seen. Bacilli coated with complement were seen sur- rounded by polymorphonuclear cells both on the surface and within superficial gastric pits. The high association of coating by IgG and IgM, along with complement activation in active chronic gastritis,

explains the polymorphonuclear neutrophil infil- tration and phagocytosis of bacilli.

The gastric mucosal lysozyme, possibly together with secretory immunoglobulins, forms part of an antibacterial defence system. Increased secretion of lysozyme has been noted in inflamed gastric m u c ~ s a . ~ ~ , ~ ' Based on the present knowledge of lysozyme function it was concluded that its presence in gastric secretions accords with the role of lyso- zyme in non-specific host defence against exogenous micro-organisms. The detection of lysozyme on the surface of C. pylori provides supportive evidence for its defensive role in gastric secretion.

Epithelial infiltration by mononuclear cells in a significantly high number of cases in the C. pylori- associated gastritis group suggests that the cellular component may have some role in the defence mechanism against C. pylori infection. Among the intraepithelial mononuclear cells were histiocytes

C. PYLORZ-ASSOCIATED CHRONIC GASTRITIS 237

and T cells. Gut mucosal lymphocytes are involved in IgA-dependent cell mediated immunity, thus aid- ing in protectin the host from infections at the

The present study shows that the cellular and humoral immune responses, except histiocytes, were significantly higher in chronic gastritis associ- ated with C. pylori infection. In vivo adsorption of host immunoglobulins IgG and IgM and the acti- vation of complement were associated with active chronic gastritis with C. pylori infection. Our find- ings provide a plausible explanation for the histo- logical features seen in chronic gastritis, and add further evidence for C. pylori having a causative role in chronic gastritis.

mucosal level. 3 2 , a

ACKNOWLEDGEMENTS

This study was supported by grants from the National University of Singapore and Jinnah Postgraduate Medical Centre, Pakistan. We thank Mr Zahid Mahmood for the statistical analysis; Messrs C. K. Ow and T. C. Tan of the National University of Singapore for technical assistance; and Ms Veronica Mok of National University of Singapore, Ejaz and Zaigham for secretarial assistance.

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