In Situ Characterization of Inflammatory Responses in the Rectal Mucosae of Patients with Shigellosis

INFECTION AND IMMUNITY,0019-9567/97/$04.0010

Feb. 1997, p. 739–749 Vol. 65, No. 2

Copyright q 1997, American Society for Microbiology

In Situ Characterization of Inflammatory Responses in theRectal Mucosae of Patients with Shigellosis

DILARA ISLAM,1,2,3* BELA VERESS,2 PRADIP KUMAR BARDHAN,4 ALF A. LINDBERG,1†AND BIRGER CHRISTENSSON2,5

Division of Clinical Bacteriology,1 Clinical Immunology,5 and Pathology,2 Department of Immunology,Microbiology, Pathology and Infectious Diseases, Karolinska Institute, Huddinge University Hospital,S-14186 Huddinge, Sweden, and Laboratory Sciences Division3 and Clinical Research Centre,4

International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh

Received 16 May 1996/Returned for modification 24 July 1996/Accepted 6 November 1996

Shigella species cause bacillary dysentery in humans by invading epithelial cells of the colonic mucosaleading to colonic epithelial cell destruction and inflammation. For further analysis of local gut inflammation,morphological changes and the potential involvement of mediators in regulatory mechanisms of cell activationand cell proliferation were studied immunohistochemically in rectal mucosal biopsies taken from patientsduring the acute phase of shigellosis and at convalescence. Rectal biopsies from 25 Shigella dysenteriae-1 and10 Shigella flexneri-infected patients and from 40 controls were studied. The frequencies of proliferative cells(Ki67-positive cells), p53-immunostaining cells, and cells coexpressing Ki67 with CD3 or with p53 wereanalyzed. Immunostaining for the inducible nitric oxide synthase (iNOS) and the endothelial NOS wasassessed. In addition, the frequencies of apoptotic cells and CD681 cells that engulf apoptotic cells wereassessed. By morphological grading, 20% of the patients had advanced inflammation (grade 3) in the acutephase; mild inflammation (grade 1) was seen in 37% of the patients at convalescence as well as in 10% of thecontrols. The findings in the present study suggest that in the acute phase of shigellosis inflammation ischaracterized by increased cell turnover in the lamina propria (LP) and the epithelium, increased iNOSexpression in the surface epithelium, and apoptosis, which seems to be associated with LP macrophages. Thefindings also suggest that neither p53 nor iNOS are important factors for the induction of apoptosis inshigellosis. Expression of p53 may be related to early cell activation in crypt epithelium. Moreover, there is anindication of an active, low-level inflammatory process at convalescence. The results thus indicate thatShigella-induced inflammation is associated with a complex series of cellular reactions in the rectal gut mucosawhich persist long after clinical symptoms have resolved.

Shigellosis is a significant cause of morbidity and mortalityparticularly of children under the age of 5 in developing coun-tries like Bangladesh (58). The active process of invasion, mul-tiplication, and spread of Shigella spp. in the mucosa causes aninflammatory enteritis (7, 31, 50). Some of the key events in thebacterial invasion process have been ascribed to the gene prod-ucts of the invasion plasmid-associated antigens (Ipa) operonof the virulence plasmid. The IpaA, -B, -C, and -D proteinsneed to be secreted to ensure epithelial adhesion, invasion, andspread to mucosal macrophages. The invasion of Shigella spp.in the gut mucosa evokes a host immune response initiated bythe activation of nonspecific inflammation. In experimentalshigellosis in monkeys, an infectious dose of 1011 CFU inducedmucosal lesions seen by endoscopy in all parts of the colon(19). In patients, the most pronounced lesions are seen in therectosigmoid area, and the intensity of the inflammation de-creases in the proximal direction (26, 57); even at autopsy theobserved mucosal inflammation was restricted to the largeintestine, and the intensity of the inflammation decreased inthe proximal direction of the colon (16). The massive killing ofmacrophages by shigellae in the lamina propria (LP) of intes-tinal villi is one of the prominent features of the infection (1).

In cell culture it has been shown that Shigella spp. can exist inphagosomes and multiply in the cytoplasm of macrophages andthat host macrophages may be killed by apoptosis, indicatingShigella spp. can trigger apoptosis (71). In general, the histol-ogy at autopsy in shigellosis shows epithelial hyperplasia, tissuedestruction, branching of crypts, and penetration of crypts intothe submucosa (16, 27, 60). In most cases the infection islimited to the intestinal mucosa, and systemic infections arerare, especially in adults. Morphologically the inflammation inshigellosis has features in common with noninfectious chronicor relapsing enteric diseases such as chronic ulcerative colitisand Crohn’s disease.Inflammation is a complex series of homeostatic reactions

involving cellular and molecular mechanisms through a net-work of cellular and mucosal signals. The inflammatory re-sponse interacts with the components of the specific immunesystem and with the repair mechanisms of the connective tissueand the mucosal epithelium. Most inflammatory diseases arecharacterized by a persistent accumulation of inflammatorycells, which is associated with chronic tissue injury. Throughtheir production and secretion of a multitude of molecules,granulocytes and activated macrophages are involved in boththe induction and the effector phase of non-antigen-specificprotection against invading organisms (9). By controlling thefunctional longevity of polymorphonuclear cells (PMN) andtheir subsequent removal by macrophages, inflammatory me-diators may play a key role in the control of inflammation (12).Cells recruited in the inflamed area may release a variety ofinflammatory mediators with both positive and negative effects

* Corresponding author. Present address: Laboratory Sciences Di-vision, International Centre for Diarrhoeal Disease Research, Bang-ladesh, GPO Box 128, Dhaka 1000, Bangladesh. Phone: 880-2-600171.Fax: 880-2-872529.† Present address: Pasteur Merieux Serums et Vaccins, 69280 Marcy

l’Etoile, France.

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on the affected area. In addition, the interplay between mac-rophage-derived cytokines and T cells also influences the ini-tiation of antigen-specific responses to microorganisms.Recently, analysis of the production of cytokines in the gut

mucosa during the acute and convalescent phases of the dis-ease has revealed a complex pattern of pro-inflammatory andTh1- and Th2-associated cytokine activity (41, 42, 44). Theseand other previous observations on the local production ofShigella-specific antibodies in the mucosa (14, 15) suggest thatin shigellosis there may be a prolonged phase of low-levelinflammation in the absence of clinical symptoms and detect-able bacteria in the stool.To further describe the dynamics of the local inflammation

in shigellosis, immunohistochemical techniques were used onearly and late rectal biopsies to analyze some parameters thatmay be important in this process: proliferation and apoptoticcell death of gut epithelium and mucosal leukocytes, the po-tential role of p53, and the possible involvement of nitric oxide(NO) as evidenced by the presence of inducible nitric oxidesynthase (iNOS).Cell kinetic studies can provide useful parameters for the

evaluation of cell activation in inflammatory responses. Theexpression of the Ki67 nucleoprotein in the G1/S/G2/M phaseof the cell cycle is widely accepted as an indicator of cellproliferation. The MIB-1 antibody recognizes both the nativeand recombinant Ki67 nuclear antigen expressed in the G1/S/G2/M phase of the cell cycle (2, 3, 6, 17, 39).The p53 nuclear phosphoprotein is best known as a negative

regulator of cell growth which interrupts progression throughthe cell cycle. In normal cells, DNA damage is associated withp53 accumulation and the switching off of cell replication,allowing extra time for repair of the genome. If the DNAdamage cannot be repaired the p53-expressing cells die byapoptosis (53, 55). Point mutation of the p53 gene is one of themost frequent genomic changes in cancer. In such cases p53proteins have a prolonged half-life leading to their accumula-tion and histochemical detection but also a loss of their inhib-itory function in cell cycle progression (3, 11, 17). However,upregulation of p53 may also be seen early in cell activation, afinding which initially led to suggestions that p53 might be anoncogene (22, 55). More recently, experimental studies in Bcells have shown p53 expression as an early event in Epstein-Barr virus (EBV)-mediated activation of B cells (61). p53 maythus be involved in the regulation of several independentphases of cell cycle regulation and apoptotic cell death.NO is a diffusible messenger with pleotropic effects in the

immune system (33). NO may act to increase or decreasecellular injury depending on the tissue involved and the phys-iological circumstance. NO production is primarily controlledby three NOSs: inducible, endothelial (eNOS), and brain(bNOS) (36). iNOS has been proposed to have a major influ-ence in the inflammatory process, and its level of expression isregulated by inflammatory cytokines (37, 40, 46, 47). Gammainterferon (IFN-g) and lipopolysaccharide (LPS) are known toinduce iNOS expression in murine macrophages (23, 67). Inhumans, iNOS is constitutively expressed apically in sinus ep-ithelium, suggesting a role for NO in the maintenance of ste-rility in the paranasal sinuses (24). NO or the products of itsredox chemistry can cause DNA damage and may result in celldeath by apoptosis or necrosis (35). It has been shown thatunder pathological conditions the proliferation of mast cells iscontrolled by iNOS, and mRNA of iNOS is strongly expressedin apoptotic cells (21). NO-mediated apoptosis has been re-ported for murine macrophages and for epithelial cells (32,62). NO may stimulate the expression of p53 prior to apopto-sis, possibly as a consequence of DNA damage (30).

Apoptosis is an intrinsic cell death program that can beactivated by a multitude of external and internal signals. Thecharacteristics of an apoptotic cell are that the chromatin iscondensed and fragmented and cytoplasmic blebs are formedresulting in small darkly stained cytoplasmic bodies (66). Itdiffers from necrosis in morphology, process of cell death, andother aspects. Activation of endonucleases in apoptosis resultsin the production of 180- to 200-bp internucleosomal DNAfragments (5, 52). The fragmented DNA can be detected withthe terminal transferase enzyme by in situ end-labeling (ISEL)of the DNA fragments, mediated by terminal deoxynucleotidyltransferase (Tdt) (8). This method preferably labels apoptoticcells and apoptotic bodies, whereas necrotic cells are identifi-able by nick translation (8).The aim of the present study was to further analyze the

dynamics of the local inflammatory reactions in the gut mucosain shigellosis. By combined morphological and (immuno-) his-tochemical techniques, proliferation and apoptotic cell deathin various compartments of the gut mucosa, including pheno-typically defined cell types, were assessed in early and laterectal biopsies. Furthermore, the potential roles of p53 andiNOS in these processes were addressed by staining for expres-sion of these molecules. These parameters were analyzed forpatient biopsies obtained during early infection with shigellosisas well as from matched controls from the same endemic area.

MATERIALS AND METHODS

Study population. Thirty-five male adult (age 20 to 45 years) patients withshigellosis, seen at the International Centre for Diarrhoeal Disease Research,Bangladesh (ICDDR,B), and 40 healthy subjects (control group) matched forsex, age, and socioeconomic status were included in the study, which was ap-proved by the Ethical Review Committee at ICDDR,B. Twenty-five of the 35patients were infected with S. dysenteriae 1 (S. dystenteria 1-infected patients[SDIP]) and 10 were infected with S. flexneri (SFIP). No other pathogenicmicroorganisms were found in stool cultures from the patients. Neither thecontrols nor the patients had experienced bloody dysentery in the 6 months priorto inclusion in the study or prior to the current infection, respectively. Onadmission, all patients exhibited clinical symptoms typical of bacillary dysenteryincluding fever, tenesmus, and blood and mucus in their feces. Most of the SDIPand a few of the SFIP had received nalidixic acid before admittance. However,the drug had been clinically ineffective and the shigellae retrieved by culture wereresistant to nalidixic acid in these cases. Patients were given 200 mg of pivme-cillinam chloride four times daily for at least one week from the day of admission.On average patients became culture negative and free of dysenteric symptomswithin 5 days after initiation of pivmecillinam treatment. Patients with additionalinfections or other major disease were not included in the study. Samples fromthis study population have been used in studies relating to other aspects of theimmune function in shigellosis.Samples and histopathological analysis. Rectal mucosal biopsies were ob-

tained during proctoscopy at two time points from each patient: (i) within 48 hof admission (after culture confirmation) and (ii) 30 to 35 days thereafter. Atconvalescence, all patients’ stool cultures were negative for infectious organisms.Rectal biopsies were obtained from controls at one time point. Biopsy specimenswere either fixed in 10% formol saline and embedded in paraffin or snap frozenin liquid nitrogen. Sections (paraffin embedded) were stained with hematoxylinand eosin for histological evaluation. To assess the degree of inflammation in thebiopsy specimens, a number of morphological parameters were documented (seeTable 1). The total score obtained by evaluating these parameters formed thebasis for grading the degree of inflammation. The scoring system followed in thisstudy is based on a scoring system used previously (45, 64).Immunohistochemistry. Immunoenzymatic staining was performed on both

serial paraffin and cryostat sections (5 mm, mounted on SuperFrost/Plus micro-scope slides; Menzl-Glaser) of the rectal mucosal biopsies. Cryostat sectionseither were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS),pH 7.4, for 5 min, washed in PBS, air dried, and kept at 2208C until being usedfor the detection of apoptotic cells or were fixed in Lana’s fixative (4% parafor-maldehyde and 3% picric acid in phosphate buffer [pH 6.9]) for 10 min, washedin PBS, air dried, and kept at 2208C until use for the detection of iNOS andeNOS.Paraffin-embedded sections were deparaffinized, rehydrated, and microwave

treated at low power for 15 min in prewarmed 10 mM citrate buffer (pH 6.0),cooled for 20 min, and then washed in water and in Hanks’ balanced salt solution(HBSS) (GibcoBRL, Life Technologies LTD, Paisley, Scotland). Prior to immu-nostaining, endogenous peroxidase activity in the paraffin and cryostat sectionswas blocked with 0.3% H2O2 in HBSS (30 min).

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For the deparaffinized, microwave-treated sections, the slides were treatedwith 10% normal goat serum (DAKO A/S, Glostrup, Denmark) to block non-specific binding. The sections were then incubated (20 h at 228C) with murinemonoclonal antibodies (MAbs) diluted in HBSS containing 1% bovine serumalbumin; this was followed by a 30-min incubation with biotinylated goat anti-mouse immunoglobulin G (IgG) (DAKO A/S) used as secondary antibody. Thesections were then treated with an avidin-biotin horseradish peroxidase complexaccording to the instructions of the manufacturer (ABC-HRP kit, DAKO A/S).A diaminobenzidine tetra hydrochloride (DAB) (0.5 g/liter) (Sigma Chemical,St. Louis, Mo.) and H2O2 (0.015%) substrate reaction was developed for 5 to 7min, and the slides were counterstained with hematoxylin and mounted. Betweenthe various incubation steps, the slides were washed with HBSS. Cell prolifera-tive activity was assessed by using the MIB-1 antibody (Immunotech, Marseille,France) to detect Ki67 antigen in the G1/S/G2/M phase, by using anti-p53 protein(DO-7; DAKO A/S), to detect wild-type and mutant p53 protein, and by usinganti-CD68 (KP1; DAKO A/S) to detect a macrophage-associated granular pro-tein. An irrelevant mouse IgG1 MAb (corresponding to the IgG subclass ofMIB-1 and KP1) and an IgG2b MAb (corresponding to DO-7), both directedagainst Aspergillus niger glucose oxidase (DAKO A/S), were used as the negativecontrols (at the same concentration as the corresponding antigen-specific MAb).For the double-staining protocol, the following rabbit antisera were used incombination with murine MAbs: anti-CD3 (affinity isolated, DAKO A/S), todetect T cells, and anti-Ki67 antigen (affinity purified, DAKO A/S). Normalrabbit immunoglobulin fraction (DAKO A/S) was used as the negative control(at the same concentration as the corresponding antigen-specific rabbit anti-serum) for the staining specificity of the rabbit antisera. The concentration ofeach MAb/antiserum was optimized by staining control samples with seriallydiluted MAb/antiserum. To avoid cross-reactivity rabbit antisera were used andthe specificity was compared with the respective MAb.In the double-staining protocols, the MIB-1 MAb was used in combination

with the anti-CD3 rabbit antiserum, the DO-7 MAb was used in combinationwith the anti-Ki67 rabbit antiserum, and ApopTag staining (Oncor, Gaithers-burg, Md.) was used in combination with the KP1 MAb. Immunoenzyme doublestainings were performed as follows: initially, after incubation with the MAb(MIB-1 or DO-7), the slides were washed and incubated with biotinylated goatanti-mouse IgG. The sections were then treated with an avidin-biotin alkalinephosphatase complex (ABC-AP kit, DAKO A/S). The alkaline phosphatasereaction was visualized with the red reaction product of the substrate kit I(Vector Laboratories Inc., Burlingame, Calif.). The sections were then blockedwith normal swine serum, followed by incubation with the second rabbit anti-serum (CD3 or Ki67) for 1 h. Then the slides were incubated with biotinylatedswine anti-rabbit IgG for 30 min as secondary antibody. The second antibody wasvisualized by using the ABC-HRP kit with the DAB reaction, and the slides werethen mounted. As controls for the double-staining procedure, instead of the firstMAb an irrelevant mouse MAb (IgG1 or IgG2b at the same concentration as thecorresponding antigen-specific MAb) was used. Also, staining with the firstantigen-specific MAb was followed with a replacement of the specific rabbitantiserum by normal rabbit immunoglobulin fraction (same concentration as theantigen-specific antiserum) to check for possible cross-staining of ABC com-plexes. In-parallel double stainings were also made in the reverse sequence.After fixation of frozen sections in Lana’s fixative, iNOS and eNOS were

identified with specific murine MAbs (diluted 1:800 in HBSS containing 2%heat-inactivated pooled human sera). The MAb detecting iNOS was raisedagainst fragments of mouse macrophage NOS which show 77% identity (84%similarity) to human hepatocyte iNOS. The MAb detecting eNOS was raisedagainst human eNOS (both MAbs were kindly provided by Jan Lundberg, Di-vision of Pharmacology, Department of Physiology and Pharmacology, Karolin-ska Institute [24]) The staining was done as described above by using ABC-HRPand the DAB substrate reaction.In paraformaldehyde-fixed frozen sections, apoptotic cells were detected

through ISEL of fragmented DNA. DNA fragments were labeled by TdT-me-diated incorporation of digoxigenin-labeled dUTP by using the ApopTag kit. Thedetection procedure of the manufacturer was followed with minor modifications.Optimal concentrations of the TdT enzyme and anti-digoxigenin-peroxidaseantibody were determined by titration. Blocking of endogenous peroxidase wasdone after the TdT treatment. The incorporated digoxygenin-labeled nucleotideswere tagged with an anti-digoxigenin antibody labeled with peroxidase, whichwas then visualized with the DAB substrate reaction (as described above). Thespecificity of the ISEL signal was confirmed by the omission of either TdT oranti-digoxygenin-peroxidase antibody in the negative controls.For the double staining ApopTag was first visualized with DAB, and then the

sections were incubated with normal goat serum followed by KP1. For KP1detection a biotinylated goat anti-mouse IgG antibody was used followed byABC-AP and the Vector red kit. In negative controls, instead of KP1 an irrele-vant mouse IgG1 MAb (at the same concentration as KP1) was used.In the LP the number of immunostained cells was counted in 5 different

high-power fields (magnification, 3400), and the average count was expressed asthe number of cells per mm2. The number of immunostained cell nuclei in thefive best oriented crypts in the crypt epithelium (CE) and in five arbitrarilydefined unit areas of the surface epithelium (SE) were counted. The number ofpositive nuclei was divided by the total number of nuclei in the crypts and in theunit areas of the SE, respectively. The average proportion (%) of positively

stained cells was calculated. For ISEL staining cell nuclei and/or apoptotic bodieswere counted as one cell if by counterstaining they were considered as part of onecellular unit.Statistical analyses. To avoid bias all evaluations were made on coded slides.

Comparisons of data between groups and at different time points were analyzedusing nonparametric statistical methods (Chi square and Mann-Whitney U-test)in the JMP statistical software package (SAS Institute, Cary, N.C.). P values of,0.05 were considered significant.

RESULTS

Histopathological evaluation. Based on an arbitrary scoringsystem the grade of inflammatory changes observed in sectionsstained with hematoxylin and eosin was estimated (Table 1).The grade of inflammation in SDIP, SFIP, and controls isshown in Table 2. In the acute phase, no difference in the gradeof inflammation was seen between SDIP and SFIP. In theacute phase, 13 of 35 patients (37%) had grade 1 inflammation,15 of 35 patients (43%) had grade 2 inflammation, and 7 of 35patients (20%) had grade 3 inflammation. Four of the 40(10%) controls had grade 1 inflammation. In contrast, at con-valescence grade 1 inflammation was significantly (P , 0.01)more frequent in SDIP (11 of 25 [44%]) than in SFIP (2 of 10[20%]).Cell proliferation in LP and mucosal epithelium. Prolifera-

tive activity, as assessed by the number of MIB-1–immunore-active cells per unit area in the LP and by the percentage of

TABLE 1. Arbitrary scoring system for grading severity ofinflammation in rectal biopsies in shigellosis

Tissue characteristic Score

Proportion of sampleswith respective tissuecharacteristic (%)

PT-S 1a PT-S 2b Control

Mucosal surfaceNormal 0 94 100 100Irregular 1 4

SEEdema 1 85 45 5Loss 2 30Erosion 3 25Ulceration 4 4Unchanged PMN 0 9 72 92Increased PMNc 1–3 91 28 8

CEUnchanged PMN 0 57 82 90Increased PMNc 1–3 43 18 10No abscess 0 90 100 100Abscess 1 10

Goblet cellsNormal count 0 40 100 100Lowered count 1 60Normal size 0 70 90 100Reduced size 1 30 10

LPUnchanged PMN 0 13 67 88Increased PMNc 1–3 87 33 12Edema 1 90 27 10Unchanged plasma cells 0 7 45Increased plasma cellsc 1–3 100 93 55Maximum total score 21

a Patient sample from the acute phase.b Patient sample at convalescence (day 30).c Arbitrary frequency estimation: 1, slight; 2, moderate; 3, high.

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MIB-1–immunoreactive cells in the SE and CE, was analyzedin biopsies from the patients and the controls (Table 3). In theacute phase the frequency of proliferative cells was signifi-cantly higher in the LP, the SE, and the CE (Fig. 1A and B)than at convalescence. The frequency of MIB-1–immunoreac-tive cells was positively correlated to the degree of inflamma-tion (Table 3). In the acute phase, there was no difference inthe frequencies of MIB-1–positive cells in SDIP and in SFIP.The 44% of SDIP that at convalescence had grade 1 inflam-mation also had a significantly higher frequency of MIB-1–immunoreactive cells than SDIP with no inflammation (datanot shown). However, in convalescence biopsies without mor-phological grade 1 inflammation, the proportion of prolifera-tive cells in the LP was significantly higher (Fig. 1C) than incontrols (Fig. 1D and Table 3). In double immunostainings forMIB-1 and anti-CD3 of acute-phase biopsies, 57% of CD31

cells in the SE were positive for MIB-1, and in the LP 10% ofthe CD31 cells were also MIB-1 positive (Fig. 1 E and F). Incontrast none of the CD31 cells in the CE were MIB-1 posi-tive.Immunohistochemical localization of p53 protein. Consis-

tent immunostaining for p53 was only seen in the CE, butoccasionally a few lymphoid cells in the LP were also p53positive. In general, the staining intensity was lower than thatof the neoplastic cells in a tumor sample serving as a positivecontrol, although the staining intensity could be quite variablefrom cell to cell in the same crypt. In the acute-phase biopsies,virtually all of the CE was stained for p53 (Fig. 2A). The p53immunoreactivity of CE cells was confirmed by the use of fivecommercially available p53-specific MAbs detecting severalunrelated epitopes and by serial titration of the DO-7 MAb

(data not shown). None of the infiltrating T cells in the cryptsexpressed detectable levels of p53. The proportion of CE cellsimmunoreactive for p53 was reduced to around 20% at con-valescence, and the staining intensity was also reduced (Table3). In controls, only rarely did one or two cells in the cryptsexpress p53 (Fig. 2B). In the CE about 60% of the p53-positivecells were also Ki67 positive (Fig. 2C). In well-oriented cryptsp53 expression was seen from the orifice of the crypt and downto the base, whereas Ki67 expression was more restricted to thelower two-thirds of the crypt.Immunohistochemical localization of iNOS and eNOS. The

presence of iNOS and eNOS in rectal mucosa biopsies wasdetected by immunostaining with specific MAbs. The stainingpatterns for iNOS and eNOS were different. The iNOS-specificMAb gave a distinct staining only in the SE, with the strongestintensity in the apical part of well-preserved SE cells (Fig. 3A).In the acute phase all biopsies were iNOS positive in the SE.At convalescence, both the staining intensity and the propor-tion of positively stained SE cells were much reduced (Fig. 3B).At convalescence, 50% of biopsies with grade 1 inflammationwere iNOS positive, and 17% of noninflamed biopsies wereiNOS positive. In the controls, only two cases had small por-tions of SE lightly stained with the iNOS MAb (Fig. 3C showsan iNOS-negative case). The expression of iNOS was neitherrelated to the infecting agent (S. dysenteriae 1 or S. flexneri) norto the degree of inflammation.The most prominent staining for eNOS was seen in the SE

and CE cells, with a granular staining mostly located in theapical part of the epithelial cells. In contrast, most of theendothelial cells in the LP appeared negative. Only endothelialcells in lymphoid plaques were strongly stained. The stainingpatterns for eNOS were similar at convalescence and in con-trols to that seen in the acute phase. However, the eNOSstaining intensity was lower at convalescence and in controls,and the distinct granular pattern was less evident (data notshown).Immunohistochemical localization of CD68. KP1 MAb was

used for the identification of macrophage-associated granularproteins in microwave-treated paraffin-embedded sections.KP1-positive (KP11) cells showed different distribution pat-terns in the acute phase and at convalescence. In the acutephase KP11 cells were mostly seen just under the SE. Atconvalescence, the KP11 cells formed clusters and oftenshowed a vesicle-like structure with an increased staining in-tensity around vesicle membranes (Fig. 4).Assessment of apoptotic cells. Intense ISEL staining was

observed in nuclei and nuclear fragments by using theApopTag kit. This staining pattern is morphologically charac-teristic of apoptotic cells and apoptotic bodies. Apoptotic bod-ies of various sizes showed distinct ISEL staining (Fig. 5A and

TABLE 2. Grading of rectal biopsies from patients and controls based on the score system for severity of inflammation

Totalscore

Inflammationgrading

No. (%) of patientsa

Acute phase Convalescent phaseControls

S. dysenteriae 1 S. flexneri S. dysenteriae 1 S. flexneri

0–2 Normal 14 (56) 8 (80) 36 (90)3–5 Grade 1 9 (36) 4 (40) 11 (44) 2 (20) 4 (10)6–8 Grade 2 11 (44) 4 (40)9$ Grade 3 5 (20)b 2 (20)c

a S. dysenteriae 1, n 5 25; S. flexneri, n 5 10; controls, n 5 40.bMean 5 13.cMean 5 11.

TABLE 3. The frequency of proliferative (MIB-1 stained) and p53-positive cells in rectal mucosal biopsy specimens from Shigella-

infected patients and controls

Samplea Inflammationgradingb

Frequency of MIB-1–stainedcells in: p53-stained

cells in CE(%)CE (%) LP

(per mm2) SE (%)

Control 0 to 1 21 6 9 41 6 16 2 6 2 2 6 2Pt S-2 0 31 6 10 133 6 35c 1 6 2 21 6 10Pt S-2 1 43 6 7c 390 6 76c 7 6 3c 23 6 7Pt S-1 1 55 6 8c 517 6 97d 11 6 5c .95Pt S-1 2 63 6 9d 723 6 79 15 6 4d .95Pt S-1 3 92 6 7d 925 6 71 21 6 7d .95

a Pt S-2, sample at convalescence; Pt S-1, sample during the acute phase.b 0, normal; 1, mild; 2, intermediate; 3, severe.c Significantly greater than control value.d Significantly greater than Pt-S 2 value for samples with grade 1 inflammation.



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B), indicating different stages of apoptosis. The cytoplasm ofapoptotic cells was also often stained, suggesting a leakage ofDNA fragments out of the nucleus. The ISEL-positive(ISEL1) cells were unevenly distributed in the mucosa. Ingeneral, the majority of the ISEL1 cells were found in theupper one-third of the LP with a patchy distribution pattern.The frequencies of ISEL1 cells in patient and control samplesare summarized in Table 4. The frequency of ISEL1 cells was

significantly higher in the acute phase than at convalescence.There was no significant difference between the frequencies ofISEL1 cells in samples with grade 1 and grade 2 inflammation,whereas samples with grade 3 inflammation had a significantlyhigher frequency of ISEL1 cells (Fig. 5A and B). In grade 3inflammation, the distribution of ISEL1 cells was more evenbut was still greater in the middle and upper part of the LP.Only in grade 3 inflammation were ISEL1 cells also observed

FIG. 1. Immunohistochemical staining of paraffin sections obtained from rectal mucosal biopsies in the acute and the convalescence phase during shigellosis andin controls. Staining of the Ki67 antigen with the MAb MIB-1 is shown in the acute phase (A and B), at convalescence (C), and in controls (D). A higher proportionof proliferative cells is seen in the SE, the LP, and the CE in the acute phase than at convalescence and in controls. Double staining in the CE (E) and SE (F) for Ki67(red) and CD3 (brown) in an acute-phase biopsy of rectal mucosa is also shown. Cells single stained for CD3 (open arrowheads) are predominant in the LP, whereascells single stained for Ki67 (closed arrowheads) are predominant in the CE. Double-stained cells (reddish brown) are infrequent and are mostly seen in the SE andthe LP (arrows). Hematoxylin was used for all counterstaining except the double staining. Magnification,3137 (panel A);3230 (panel B);392 (panels C and D);3155(panels E and F).



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in the CE and the SE. In control samples very few ISEL1 cellswere identified (Fig. 5C). In the acute phase the frequency ofdouble-positive cells (ISEL and KP1) was high (Table 4 andFig. 5A and B), whereas at convalescence and in controls, onlya few ISEL1 cells were also CD68 positive (Fig. 5C).

DISCUSSION

This study provides new information on the dynamics of thelocal inflammatory process in the gut during shigellosis. Wedescribe the processes of cell proliferation and apoptotic celldeath in the target tissue and in phenotypically defined leuko-cyte subsets during shigellosis. The potential role of p53 in celldeath and proliferation, as well as the potential role of NO asan inflammatory mediator, was also addressed.Previous studies of the rectal mucosa in shigellosis have

mostly been concerned with histopathological findings (4, 16,26, 28, 48). In this study we have shown by morphologicalcharacteristics that a prolonged inflammation is more frequentin patients infected with S. dysenteriae 1 than in patients in-fected with S. flexneri.The expression of the p53 protein in infectious colitis has not

previously been reported. Somewhat unexpectedly, p53 expres-sion was strongly associated with Ki67 (MIB-1) expression inthe acute phase and, to a lesser extent, at convalescence. Thisfinding indicates that induction of the regenerative process ofthe mucosal epithelium is initiated early on in the disease andcontinues long after resolution of clinical symptoms. It is notclear if epithelial regeneration (antigen specific and nonspe-cific) is already initiated at the onset of disease since the earlybiopsies were taken at the admission to the hospital, whichcould be several days after the onset of clinical symptoms.These findings are in accordance with previous morphological

studies of shigellosis which show increased mitotic rates in theCE at sites distant from the ulcers (16).p53 upregulation delays cell cycle progression in normal

cells with damaged DNA structure, allowing DNA repair be-fore DNA replication takes place. Inhibition of the normal p53function may be important in cancer through mutation andoverexpression (10, 34, 49) and in viral infections where for-mation of complexes between p53 and SV40LT, papillomavirus E7 and E6, or adenoma viral E1A and E1B inhibits itsnormal function, thereby leading to an amplification of virus-infected cells (22, 38, 51, 65). However, both earlier and morerecent studies on p53 expression after EBV stimulation suggestthat normal p53 may be upregulated very early in cell activa-tion preceding S phase entry (18, 61). Moreover, p53 overex-pression has also been reported in benign lesions in the skin, indermatofibromas, in pleomorphic adenomas, and in colorectaladenomas (in CE cells) where p53 accumulation was associ-ated with cell proliferation (54–56, 68).Thus the gut epithelial p53 upregulation in shigellosis may

not be specific for this infection but rather reflect stressfulinfluences by the inflammation blocking the cell cycle progres-sion of cells that have already entered the G1 phase, allowingfor cellular repair before cell cycle progression. However, assuggested by the in vitro findings in EBV-stimulated B cells,this p53 upregulation could also reflect a specific p53 functionin early cell activation. The possibility that the accumulation ofp53 protein would reflect activation of an apoptotic pathwayseems less likely, since the number of apoptotic cells in the CEwas generally lower than in the SE or LP, where p53-express-ing cells were rare. Information on the relationship betweencell cycle stage and apoptosis in vivo is, however, as yet quitemeager.In the acute phase, superficial ulcerations were seen only in

FIG. 2. Immunohistochemical localization of the p53 antigen with the DO-7 MAb in rectal mucosal biopsies. In the acute phase (A) positive staining is seen invirtually all nuclei of the CE cells (arrow) but with a variable, beige to brown, staining intensity. Very few nuclei in the LP are positive (open arrowhead). In a controlsample (B) very few positive cells are seen (arrow). In the acute phase (C) a double staining for p53 (red) and Ki67 (brown) shows that most CE cell nuclei are doublepositive for p53 with Ki67 (arrow). Only a few CE cell nuclei appear positive for p53 (closed arrowhead). A few lymphoid cell nuclei in the LP are positive for Ki67only (open arrowhead). Hematoxylin counterstaining, except in double-stained sample. Magnification, 395 (panel A); 3161 (panel B); 3239 (panel C).



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FIG. 3. Expression of iNOS in cryostat sections of rectal mucosa. In the acute phase (A) iNOS staining is restricted to the SE, at convalescence (B) very few SEcells show iNOS expression along the apical aspect of the cytoplasm (arrow), and virtually no iNOS staining was seen in control biopsies (C). Hematoxylincounterstaining. Magnification, 3187.

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3 of the 7 cases with grade 3 inflammation. Most of the biopsysamples had erosions on the SE. In previous studies on gutinflammatory changes in shigellosis, the distinction of necroticversus apoptotic cell death was not thoroughly analyzed (16,19). Our morphological findings and the ISEL staining whichpreferentially labels apoptotic and not necrotic cells (8) indi-cate that the observed cellular destruction was due to apoptosisand that the apoptotic bodies were cleared by phagocytosis.The period in which DNA fragmentation and the first mor-phological changes occur is extremely short compared to thetotal life span of the cells. Also the onset of morphologicalchanges occurs simultaneously with or may even precede DNAfragmentation (25, 29). In addition to CD681 macrophagesengulfing ISEL1 apoptotic cells, macrophages themselves maybecome apoptotic, especially in cases of severe acute shigello-sis. Although at convalescence the frequency of apoptotic cellswas markedly reduced, still a few macrophages with apoptoticbodies were seen. Considering the rapid turnover of apoptoticbodies in macrophages this finding is compatible with the no-tion that in shigellosis there is a low level of persistent inflam-mation in the gut well after the disease has been clinicallyresolved.It has recently been shown that secreted IpaB is essential for

S. flexneri to induce apoptosis in macrophages (70). The mech-anisms for apoptosis induction in shigellosis may be several: (i)direct generation of second messengers that start apoptosis,(ii) secretion of cytotoxic molecules which induce apoptosis,and (iii) interference with a putative macrophage factor thatcontinuously inhibits the cell apoptotic machinery (70). In an-imal studies it has been shown that S. flexneri induces apoptosisin peritoneal macrophages by releasing interleukin-1, which inturn mediates an early inflammatory response in epithelial

tissue (69). Also in the early phase of shigellosis, the localproduction of pro-inflammatory cytokines has been shown tobe maximal (42). Possibly, cytokines also contribute to theapoptotic death of several leukocyte subsets, including T cells.Further elucidation of the mechanisms and control of apopto-sis in shigellosis will provide information in the pathophysiol-ogy of inflammation in this disease.NO-dependent processes may influence immune and inflam-

matory mechanisms. Pro-inflammatory cytokines may induceNO production in human endothelial and epithelial cells (37,40, 46, 47). We found an induced epithelial iNOS expression inthe SE of the gut in the acute phase of shigellosis. This may berelated to Shigella-derived factors, possibly LPS, since in amurine model system LPS together with IFN-g are the majorinducers of iNOS in macrophages (23, 35, 67). However, inparallel studies (42–44) mucosal levels of IFN-g and IFN-greceptors were lower in the acute phase than in the convales-cent phase of the disease, indicating that the level of IFN-gmay be inversely correlated to the iNOS expression pattern.Other host-derived inflammatory mediators may induce LPS-mediated iNOS expression in humans. Biopsies taken at theconvalescent phase had a weak immunostaining for iNOS,although the normal gut flora was not able to induce a detect-able level of iNOS in controls. This suggests that even 30 daysafter the onset of the disease an active low-level inflammatoryprocess is still persistent.In the present study p53, iNOS, and apoptotic cells were not

colocalized. p53 was seen virtually only in the proliferative cellsof the CE while iNOS was localized in the SE and apoptoticcells were localized in the LP, suggesting that there is no directlink between iNOS and p53 expression and apoptotic celldeath in shigellosis. We assume that in shigellosis the invasion

FIG. 4. Immunohistochemical localization of CD68-expressing cells stained with the KP1 MAb. In the acute phase (A) the immunoreactive cells are mostlyscattered under the SE. At convalescence (B) a distinct pattern is seen with clusters of larger, more intensely stained cells preferentially located deeper in the LP.Hematoxylin counterstaining. Magnification, 3187.



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FIG. 5. Identification with the ApopTag kit of apoptotic cells in an acute-phase rectal biopsy sample with grade 3 inflammation (A and B). Brown apoptotic cellsand bodies are seen in the SE, the LP, and the CE. Red CD68-expressing cells are identified mainly in the subepithelial area of the LP. Two patterns of ApopTag-and CD68-positive cells are seen. Some cells are double positive, with a condensed brown nuclear staining and red cytoplasm (panel B, open arrowhead). Other cellscontain only brown apoptotic bodies in their cytoplasm (panel B, arrow). A double-stained control biopsy (C) shows red cytoplasmic staining of CD68-expressing cells,and only one or two brown apoptotic cells are seen (arrow). Magnification, 3187 (panels A and C); 3325 (panel B).

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of microorganisms is the principal factor initiating both anti-gen-specific immune responses and nonspecific inflammatoryresponses. Since at convalescence no culture-positive patho-gens were found in feces, the continuous inflammatory re-sponses could be due either to intracellular sequestration of alow number of viable Shigella sp. or to bacterial components.However, immunohistochemical staining with MAb MASFB(specific for the O antigen of S. flexneri and S. dysenteriae 1 [13,19]) did not convincingly show the presence of bacteria inapoptotic macrophages (data not shown). Alternatively, apostinfectious autoimmune-like reaction, possibly as a conse-quence of a breaching of self tolerance associated with theimmune response to Shigella infection, could also explain thepersistence of inflammation at convalescence. Many similari-ties between the response patterns in shigellosis and in inflam-matory bowel diseases would fit well into such a concept. Re-active arthritis (ReA) is sometimes seen following entericinfections by Yersinia spp., and it is sometimes seen after shig-ellosis due to infection with S. flexneri carrying the 2-Md plas-mid, pHS-2 (20, 59, 63). This plasmid encodes an HLA-B27mimetic epitope that may be involved in the development ofReA (59). Since ReA was not seen in the patients of this studyduring the 30-day follow-up period, ReA could not be corre-lated to disease severity or persistent gut inflammation.Our data suggest that in acute shigellosis, there is an early

induction of epithelial regeneration, and this proliferative re-sponse is associated with p53 activation. Furthermore, apopto-sis seems to be a major pathway for leukocyte death in the LP.The uncoupling of the staining patterns for iNOS, p53, andapoptosis seems to suggest that neither iNOS nor p53 areimportant factors for the induction of apoptosis. The persis-tence of p53, Ki67, and iNOS expression in convalescent bio-psies indicates that an active, but low-level, inflammatory pro-cess may continue in clinically cured patients. Whether thischronic phase is due to incomplete eradication of the infectionor is a postinfectious autoimmune reaction reminiscent of thatseen in chronic bowel inflammation remains unclear.

ACKNOWLEDGMENTS

We are indebted to the individuals with shigellosis and the healthyindividuals from Bangladesh for their participation in the study. Wethank Pradip Chandra Dash for his assistance in recruiting patientsand control subjects. We also thank Birgitta Axelsson (Division ofPathology, Department of Immunology, Microbiology, Pathology andInfectious Diseases, Karolinska Institute, Huddinge University Hospi-tal) for technical assistance.This study was supported by a grant from the Swedish Agency for

Research Co-operation with Developing Countries (SAREC); ICD-

DR,B; the Swedish Medical Research Council (Grant no. 16x 656);and funds of the Karolinska Institute.

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TABLE 4. The frequency of ISEL1 cells in rectal mucosal biopsyspecimens from Shigella-infected patients and controls

Samplea Inflammationgradingb

Frequency of positive stainedcells in: KP11 and ISEL1

cells in LP(per mm2)CE

(%)LP

(per mm2)SE(%)

Control 0 to 1 0 86 3 0 4 6 3Pt S-1 1 to 2 0 656 8c 4 6 3 15 6 3Pt S-1 3 2–4 2056 36d 12 6 4 56 6 15Pt S-2 0 to 1 0 276 4e 0 5 6 3

a Pt S-2, convalescent phase; Pt S-1, acute phase sample.b 0, normal; 1, mild; 2, intermediate; 3, severe.c Significantly greater than Pt-S 2 value.d Significantly greater than Pt-S 1 value for samples with grade 1 to 2, inflam-

mation.e Significantly greater than control value.



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In Situ Characterization of Inflammatory Responses in the Rectal Mucosae of Patients with Shigellosis

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