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The Journal of Immunology, 2006, 177: 2015-2022.
Copyright © 2006 by The American Association of Immunologists

Reduced Peripheral and Mucosal Tropheryma whipplei-Specific Th1 Response in Patients with Whipple’s Disease1

Verena Moos2,*, Désirée Kunkel*, Thomas Marth{dagger}, Gerhard E. Feurle{ddagger}, Bernard LaScola§, Ralf Ignatius, Martin Zeitz* and Thomas Schneider*

* Medizinische Klinik I, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; {dagger} St. Josefs Krankenhaus, Zell/Mosel, Germany; {ddagger} Deutsches Rotes Kreuz Krankenhaus, Neuwied, Germany; § Unité de Rickettsies, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6020, Faculté de Medicine, Marseille, France; and Abteilung für Medizinische Mikrobiologie und Infektions-Immunologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Whipple’s disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple’s disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple’s disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-MarseilleT has enabled the first analysis of T. whipplei-specific reactivity of CD4+ T cells. A robust T. whipplei-specific CD4+ Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple’s disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple’s disease patients and may contribute to the fatal natural course of the disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Whipple’s disease, first described by George Hoyt Whipple in 1907 (1), is an infectious disease caused by the actinomycete Tropheryma whipplei (2). The most frequent manifestations of Whipple’s disease are weight loss, diarrhea, polyarthralgia, fever, lymphadenopathy, and cardiac and CNS symptoms (3). The course of Whipple’s disease is fatal, unless treatment with antibiotics is initiated (4).

T. whipplei is assumed to be a microorganism present in the environmental soil and water (5). An oral route of acquisition was proposed (6), and in recent studies, T. whipplei DNA was detected in the saliva (7), marginal and subgingival plaque (8), and feces (9) of healthy subjects in which T. whipplei-specific IgG Abs were identified in over 70% of the cases (10). Whipple’s disease is very rare despite the almost ubiquitous occurrence of T. whipplei in the environment, suggesting that host factors are necessary to permit an infection.

In patients with Whipple’s disease, cutaneous responses to recall Ags and peripheral T cell proliferation are reduced after stimulation with PHA, Con A, and Abs to CD2. These functions improve somewhat during treatment, but remain impaired even in long-standing remission (11, 12, 13, 14). Apparently, the Th1 reactivity in the periphery and the intestinal mucosa is impaired (14). Low serum concentrations of IL-12p40, a reduced production of IL-12 in monocytes (15, 16), and the presence of M2/alternatively activated macrophages that favor the development of Th2 responses in the intestine (17) may explain these impaired Th1 cell functions. Despite reduced Th1 activity, general immunocompetence seems to be unaffected, as no systemic opportunistic infections have been reported in Whipple’s disease patients. Previous investigations focused on unspecific reactivity of lymphocytes and thus could not contribute to clarify this apparent discrepancy. Therefore, the investigation of T. whipplei-specific immune responses, made possible through the recently established cultivation of T. whipplei, appears particularly important. Consequently, we used T. whipplei lysate to investigate specific reactivity of peripheral and duodenal CD4+ T cells in patients with Whipple’s disease and control subjects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and control subjects

We studied CD4+ T cell reactivity in 32 patients with different stages of Whipple’s disease (13) (Table I, 24 males (M), 8 females (F), mean age, 57.2; range, 41–84 years), compared with control groups without evidence of disease: I, 16 young subjects (8 M, 8 F, mean age, 30.3; range, 21–39); II, 27 elderly subjects, age-matched to Whipple’s disease patients (16 M, 11 F, mean age, 54.9; range, 41–88); and III, 11 active triathletes, age-matched to young subjects (11 M, mean age, 32.7; range, 25–43). Triathletes practice swimming in the river Neckar in Heidelberg where T. whipplei was detected in sewage plants (5) and thus are supposed to have enhanced contact to environmental T. whipplei. As disease-control, 17 patients with active tuberculosis (TB)3 were selected for group IV (10 M, 7 F, mean age, 48. 9; range, 23–85; 9 with pulmonary, 6 with lymph node, and 1 with bone and ovarian TB, respectively). The diagnosis was based on the detection of Mycobacterium tuberculosis by Ziehl-Neelsen staining and cultivation. TB patients were selected for positive tuberculin skin test, and tuberculin-specific Th1 reactivity (IFN-{gamma} expression after stimulation; see below).


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  		Table I. Patient characteristics

 
Duodenal biopsies were obtained of 15 Whipple’s disease patients (11 M, 4 F, mean age, 57.6; range, 41–74) (Table I) and in 11 age-matched subjects during follow-up of gastric ulcer disease (Table II 8 M, 3 F, mean age 67.3; range 43–88; no duodenal involvement, Helicobacter pylori-negative at sampling). Permission for the study was obtained from the clinical ethics committee of the Charité and all subjects gave their written consent to participate.


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  		Table II. Characteristics of control subjects for DL stimulationa

 
Cell culture

Cell cultures were grown in RPMI 1640 with Glutamax (Invitrogen Life Technologies) supplemented with penicillin/streptomycin (100 U/100 µg/ml; Biochrom), and 2-ME (50 µM; Invitrogen Life Technologies) (culture medium) at 37°C and 5% CO2. FCS (Sigma-Aldrich) or autologous serum was added as indicated.

Isolation of PBMC and duodenal lymphocytes

Blood was collected in sodium-heparinized tubes (Vacutainer; BD Biosciences), and PBMC were separated by Ficoll-Hypaque-gradient (Pharmacia).

Duodenal lymphocytes were isolated from six to eight biopsies as described (18). In short, minced biopsies were digested for 3 h on a shaker in culture medium supplemented with 10% FCS, HEPES (25 mM; Invitrogen Life Technologies), amphotericin (250 µg/ml; PAA Laboratories), trypsin inhibitor (100 µg/ml; Sigma-Aldrich), DNase (100 µg/ml; Roche), and collagenase type CLS III (300 µg/ml; Biochrom). The suspension was filtered through a mesh, followed by a cell strainer (70 µm; BD Biosciences), and washed twice in PBS containing 0.5% BSA (Sigma-Aldrich). Viability of all cell preparations was >90%.

CFSE labeling

PBMC were washed twice in PBS, suspended at 1 x 107 cells/ml in PBS containing CFSE (0.5 µM/ml; Molecular Probes), incubated for 3 min at room temperature, and washed two times with culture medium (10% FCS).

Stimulation of PBMC, duodenal lymphocytes, and whole blood

Ag-specific effector T cells were determined through short-term (6–12 h) stimulation (19). Brefeldin A (10 µg/ml; Sigma-Aldrich) was added for the last 3 h to assess cytokine production. All preparations were washed and resuspended after fixation in PBS/0.5%BSA/0.02%NaN3 (PBA), and stored at 4°C.

Duodenal lymphocytes and PBMC were stimulated in culture medium (5% FCS) at 106 cells/ml in the presence of anti-CD28 (1 µg/ml, clone CD28.2, low endotoxin, no NaN3, BD Biosciences) for 12 h, and fixed with 4% formalin in PBS. For stimulation of duodenal lymphocytes, autologous CFDA-labeled PBMC were added at a ratio of 1:2.

Fresh heparinized blood (500 µl) was stimulated in 15-ml polypropylene tubes (Eppendorf) in the presence of anti-CD28 and anti-CD49d (20) at 2 µg/ml (clone 9F10, low endotoxin, no NaN3; BD Biosciences) for 6 h. At the end of incubation, 50 µl of 20 mM EDTA (pH 7.5) was added, incubated for 10 min at room temperature and mixed vigorously. Nine volumes of FACS-lysing solution (BD Biosciences) were added for 15 min at room temperature for lysis of erythrocytes and fixation.

Tuberculin (6 U/ml; Chiron Behring), lysate of CMV (6 µg/ml purified CMV grown on human fibroblasts; Biodesign), tetanus toxoid (10 µg/ml; Aventis), lysates of heat-inactivated Giardia lamblia trophozoites (5 x 104/ml; Seramun Diagnostica), and actinomycetes closely related to T. whipplei (21) (Cellulomonas hominis, and Cellulosimicrobium cellulans (synonym: Cellulomonas cellulans), each at 107 bacteria/ml) were used as control Ags.

Staphylococcus enterotoxin B (SEB) (2 µg/ml; Sigma-Aldrich) served as positive control. Negative controls contained no supplements (for SEB), and anti-CD28, or anti-CD28 and anti-CD49d (for Ag-specific stimulations), respectively, and were subtracted from the values obtained after stimulations. Values after stimulation below background level were defined as 0.

T. whipplei-specific stimulations

T. whipplei-specific stimulations were established in 10 healthy subjects with heat-inactivated, sonicated T. whipplei Twist-MarseilleT cultivated in confluent MRC5 fibroblasts (ATCC number CCL-171; American Type Culture Collection) (10, 22). The concentration in the preparation was ~109 T. whipplei/ml (determined by quantitative PCR of 16S rDNA (22)) and 104 MRC5/ml (determined from a confluent monolayer of MRC5). Lysate was titrated and a dilution of 1/100 (107 bacteria/ml and 102 MRC5/ml) induced highest cytokine production in PBMC (data not shown). Lysate of uninfected MRC5 (102 cells/ml; provided by Dr. H. W. Mittrücker, Max-Planck Institute for Infection Biology, Berlin, Germany) was used as negative control and lysate of cell-free grown T. whipplei (107 bacteria/ml) (23) to exclude host factors of infected MRC5.

Generation of short-term T cell lines

CD4+ T cell lines were generated as described previously (24). Briefly, 106 PBMC/ml in culture medium (5% autologous serum) were stimulated with T. whipplei lysate (107 bacteria/ml). Subsequently, IL-2 (20 U/ml; Proleukin S; Chiron-Behring) was added on days 2, 5, and 9. On day 14, cell lines and autologous CFDA-labeled PBMC (1:4, 106 cells/ml) were restimulated as described for PBMC for 6 h.

Blocking of endotoxins with polymyxin B

Polymyxin B was added at 2 µg/ml to stimulations of PBMC of healthy subjects with LPS (0.1 mg/ml; Sigma-Aldrich) and T. whipplei lysate (107 bacteria/ml).

Flow cytometric analysis

Ag-specific CD4+ T cells were analyzed by four-color FACS analysis (19). Cell preparations were washed in PBA, blocked at room temperature 10 min in 50 µl of PBA containing 2% Beriglobin (Behring), then the Abs were added in 50 µl of PBA in dilutions determined before use (data not shown) for 15 min at room temperature. Cells were washed and resuspended in PBA for analysis. Intracellular staining (IFN-{gamma}, IL-2, IL-10, TNF-{alpha}) was performed in the presence of 0.5% saponin (Sigma-Aldrich). Data were acquired on a FACSCalibur (BD Biosciences), and collected and analyzed with CellQuest software (BD Biosciences).

Gates were set on lymphocytes on a sideward-/forward-scatter dot blot, and on CD4+ cells. At least 50,000 CD4+ peripheral and 10,000 CD4+ duodenal lymphocytes were analyzed. The following Abs were used: anti-CD3 (UCHT1), anti-CD4 (SK3), anti-CD154 (CD40L, TRAP1), anti-IFN-{gamma} (B27), anti-TNF-{alpha} (Mab11), anti-IL-2 (MQ1–17H12), and anti-IL-10 (JES3-19F1) from BD Biosciences; anti-CD4 (MT310), anti-CD25 (ACT-1), anti-CD69 (FN50) from DakoCytomation; and anti-CD69 (TP1.55.3), and anti-HLA-DR (Immu357) from Caltag Laboratories. Mouse IgG1, mouse IgG2b, and rat IgG2 (all BD Biosciences) served as isotype controls.

Statistical analysis

Data were analyzed by means of the Kruskal-Wallis test. Mann-Whitney’s comparison test was used for post hoc analysis. Values of p <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Establishing a T. whipplei-specific stimulation

Optimal Ag dose. Lysate from T. whipplei-infected MRC5 fibroblasts, uninfected MRC5, and SEB were used to stimulate PBMC in fresh whole blood of healthy subjects. The stimulation revealed a dose-dependent increase in the percentage of IFN-{gamma}+cells of CD4+ T cells, and virtually all IFN-{gamma}+ cells expressed CD69. A maximal cytokine response was achieved with 107 bacteria/ml, subsequently used for additional experiments (Fig. 1a).


Figure 1
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  		FIGURE 1. Establishing a T. whipplei-specific stimulation in healthy subjects. a and b, One representative example of 10 (a) respective 3 (b) experiments. a, Stimulation of whole blood with SEB, lysate of MRC5, and lysate of T. whipplei-infected MRC5 (106 or 107 bacteria/ml). b, Stimulation of a T. whipplei-specific short-term T cell line with SEB and T. whipplei lysate (107 bacteria/ml). c, Stimulation of PBMC in the presence of polymyxin B (PB) (n = 5), and with proteinase K-digested (Pkdig) T. whipplei lysate (n = 10). The percentage of CD69+IFN-{gamma}+ cells of CD4+ lymphocytes stimulated without PB and with undigested T. whipplei lysate was set as 100%. Median and SD are shown.

 
Short-term T cell line. T. whipplei-specific, short-term T cell lines were established from three healthy subjects to confirm Ag specificity of the reaction. The frequency of T. whipplei-specific CD69+IFN-{gamma}+ cells of CD4+ lymphocytes increased after re-stimulation with 107 bacteria/ml at day 14 (Fig. 1b). The reactivity to MRC5 (data not shown) and SEB remained unchanged (Fig. 1b).

Specificity of T. whipplei-specific stimulation. Polymyxin B was added to stimulations of healthy subjects to preclude that remnants of endotoxins in the T. whipplei lysate induce the reactivity. Polymyxin B reduced the frequency of CD69+IFN-{gamma}+ cells of CD4+ lymphocytes reacting against LPS to 64 ± 8.3% whereas the T. whipplei-specific Th1 reactivity was not affected (Fig. 1c).

To show that proteins or peptides contained in the lysate induce the observed Th1 reactivity, PBMC of 10 healthy subjects were stimulated with proteinase K-digested T. whipplei lysate. The stimulation with digested lysate caused loss of Th1 responses (Fig. 1c).

T. whipplei-specific CD4+ T cell reactivity in Whipple’s disease patients compared with healthy and disease control subjects

Peripheral blood. T. whipplei-specific T cell responses in whole blood of 32 mainly middle aged patients with different stages of Whipple’s disease (Table I) were compared with those of three groups of healthy subjects (young subjects, age-matched subjects, and active triathletes), and to those of patients with TB. Patients with Whipple’s disease revealed a significantly reduced percentage of T. whipplei-specific IFN-{gamma}-producing CD4+ T cells compared with the four control groups (Fig. 2a). The frequency of T. whipplei-specific CD69+IFN-{gamma}+ cells of CD4+ lymphocytes was highest in the group of triathletes, followed by the age-matched subjects and the young subjects. TB patients revealed the lowest T. whipplei-specific reactivity of the control groups. The reactivity of TB patients was still significantly higher than in Whipple’s disease patients but significantly lower than in triathletes and age-matched controls (Fig. 2a).


Figure 2
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  		FIGURE 2. T. whipplei-specific stimulation (107 bacteria/ml) of whole blood and duodenal lymphocytes. Individual percentages of CD69+IFN-{gamma}+ cells of CD4+ lymphocytes and median are shown. a, Whole blood. b, Duodenal lymphocytes. c, T. whipplei-specific reactivity in different stages of Whipple’s disease (WD). d, Time course of T. whipplei-specific reactivity of single patients. Cyoung, young healthy subjects; Tri, healthy triathletes; Cage-matched, age-matched healthy subjects; WD, WD patients; TB, TB patients; (I->II), status changed from active disease (I) to remission under treatment (II); (II->III), status changed from remission under treatment (II) to sustained remission (III).

 
The individual frequencies of CD69+IFN-{gamma}+ cells of CD4+ T cells did not differ using different lysates of cell-free grown T. whipplei (107 bacteria/ml) to stimulate PBMC of 10 patients with Whipple’s disease and 10 healthy subjects (data not shown).

Duodenal mucosa. The percentage of T. whipplei-specific CD4+ duodenal lymphocytes expressing IFN-{gamma} was significantly lower in patients with Whipple’s disease compared with age-matched subjects (Table II) (Fig. 2b).

Subgroup analysis of patients with Whipple’s disease. Because the proliferative capacity of T cells seems to recover after treatment, we considered the development of T. whipplei-specific reactivity in the course of Whipple’s disease. The percentages of T. whipplei-specific Th1 cells did not differ between patients with active Whipple’s disease, remission under treatment, and sustained remission (Fig. 2c). In addition, the individual reactivity of patients tested at two to three different time points representing distinct disease activities had no major variations (Fig. 2d).

CD4+ T cell reactivity to other bacterial and viral Ags

CD4+ T cell reactivity to tetanus toxoid, tuberculin, actinomycetes, G. lamblia, and CMV. Common Ags were used to investigate the general Ag-specific reactivity of Whipple’s disease patients. A similar proportion of patients, age-matched subjects, and TB patients showed Th1 reactivity to CMV and tetanus toxoid (Fig. 3a). Additionally, the frequencies of IFN-{gamma}+ CMV- and tetanus toxoid-specific CD4+ T cells in these three groups were similar (Fig. 3a).


Figure 3
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  		FIGURE 3. Stimulation of whole blood. a, CMV-lysate, tuberculin, and tetanus toxoid. b, C. hominis, C. cellulans (107 bacteria/ml), and G. lamblia trophozoites (5 x 104/ml). Median and SD of persons with an Ag-specific reactivity of >0.03% CD69+IFN-{gamma}+ cells respective 0.02% CD154+(CD40L)IFN-{gamma}+ cells of CD4+ lymphocytes are shown. [%]reacting, Percent of persons with reactivity.

 
Obviously, all of the TB patients revealed reactivity to tuberculin whereas only 36% of patients with Whipple’s disease, and 44% of age-matched subjects showed a reaction. As expected, TB patients revealed a significantly higher frequency of tuberculin-specific CD69+IFN-{gamma}+ cells of CD4+ T cells compared with Whipple’s disease patients as well as age-matched subjects (Fig. 3).

Because giardiasis might be associated with Whipple’s disease (25), lysates of G. lamblia trophozoites were used as additional stimulus and stimulation with apathogenic actinomycetes was performed to exclude cross-reactivity being responsible for the observed reactivity to T. whipplei. The reactivity (percent of CD154 (CD40L)+IFN-{gamma}+cells of CD4+lymphocytes) and the proportion of individuals reacting to G. lamblia trophozoites, and the actinomycetes C. hominis and C. cellulans closest related to T. whipplei were similar in patients and healthy subjects (Fig. 3b).

CD4+ T cell reactivity to SEB. The percentage of peripheral CD69+IFN-{gamma}+ cells of CD4+ T cells of patients with Whipple’s disease reacting to the superantigen SEB was similar compared with the four control groups (Fig. 4a). In addition to intracellular expression of IFN-{gamma}, the percentage of CD4+ T cells expressing IL-2, IL-10, and TNF-{alpha} was determined in 14 Whipple’s disease patients and 9 age-matched subjects after SEB-stimulation. No significant differences in the percentage of CD4+ T cells expressing IL-2 (Whipple’s disease: 5,03 ± 3,94, age-matched subjects: 4.09 ± 3.27% IL-2+ cells of CD4+ T cells), and TNF-{alpha} (Whipple’s disease: 8.19 ± 4.94, age-matched subjects: 8.29 ± 5.83% TNF-{alpha}+ cells of CD4+ T cells) were detected. IL-10 was detected only in a low percentage of CD4+ cells compared with IL-2 and TNF-{alpha}. However, IL-10-expression was similar in Whipple’s disease patients and control subjects (Whipple’s disease: 0.29 ± 0.22, age-matched subjects: 0.42 ± 0.27% IL-10+ cells of CD4+ T cells).


Figure 4
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  		FIGURE 4. SEB stimulation of whole blood and duodenal lymphocytes. Individual percentages of CD69+IFN-{gamma}+ cells of CD4+ lymphocytes and median are shown. a, Whole blood. b, Duodenal lymphocytes. Cyoung, young subjects; Tri, triathletes; Cage-matched, age-matched subjects; WD, WD patients; TB, TB patients.

 
SEB stimulation of duodenal lymphocytes resulted in a significantly higher percentage of IFN-{gamma}-expressing CD69+ cells of CD4+ lymphocytes in patients with Whipple’s disease than in age-matched subjects (Fig. 4b).

Activation status of CD4+ PBMC after T. whipplei-specific and SEB stimulation

The expression of the activation markers CD69 and CD154 was investigated to exclude that repressed IFN-{gamma} expression causes the reduced reactivity to T. whipplei. The percentage of peripheral CD4+ T cells of patients with Whipple’s disease expressing CD69 and CD154 was reduced compared with all control groups after stimulation with T. whipplei lysates (Fig. 5, a and b). As for IFN-{gamma} expression, the percentage of CD69+ cells of CD4+ lymphocytes of TB patients was significantly reduced compared with the two groups of healthy subjects (Fig. 5a). In contrast, stimulation with SEB resulted in similar percentages of CD4+ T cells expressing CD69 and CD154 in Whipple’s disease patients and control subjects (Fig. 5, c and d).


Figure 5
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  		FIGURE 5. Activation of CD4+ lymphocytes after T. whipplei-specific and SEB stimulation of whole blood. Individual percentages and median are shown. a and b, Stimulation with T. whipplei and the percentage of (a) CD69+ and (b) CD154+ cells of CD4+ lymphocytes, respectively. c and d, SEB stimulation and the percentage of (c) CD69+ and (d) CD154+ cells of CD4+ lymphocytes. Cyoung, young subjects; Cage-matched, age-matched subjects; WD, WD patients; TB, TB patients.

 
Influence of IL-2 addition to T. whipplei-specific stimulation

To overcome a possible anergy of T cells, whole blood from healthy subjects and Whipple’s disease patients was stimulated with T. whipplei lysate, either alone or in the presence of a high dose of exogenous IL-2 (50 U/ml). The addition of IL-2 did not significantly enhance the percentage of CD69+ of CD4+ T cells expressing IFN{gamma} (Fig. 6). In healthy subjects, only CD25+ of CD4+ T cells could be significantly stimulated by IL-2, while in patients neither CD69+ nor CD25+ of CD4+ T cells could be stimulated to produce additional IFN-{gamma}.


Figure 6
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  		FIGURE 6. Addition of exogenous IL-2 to T. whipplei-specific stimulations. Whole blood of healthy subjects (n = 4) and WD patients (n = 9) was stimulated with T. whipplei with or without IL-2 (50 U/ml). Median and SD of the percentage of IFN-{gamma}+ cells of CD4+ lymphocytes are shown.

 
Activation status of freshly isolated PBMC and duodenal lymphocytes

Patients with Whipple’s disease showed a significantly increased percentage of activated (CD4+CD25+, CD3+HLA-DR+) duodenal and peripheral lymphocytes compared with healthy age-matched subjects (Fig. 7).


Figure 7
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  		FIGURE 7. Expression of CD25 and MHC II (HLADR) on freshly isolated duodenal and peripheral lymphocytes. Individual percentages and median are shown. a, The percent of CD25+ cells of CD4+CD3+ lymphocytes. b, The percentage of HLADR+ cells of CD3+ lymphocytes. Cage-matched, age-matched subjects; WD, WD patients.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we found that the Th1 response of peripheral and mucosal effector CD4+ T cells of healthy individuals was consistent and dose dependent when stimulated with a lysate of T. whipplei whereas this T. whipplei-specific Th1 response was significantly reduced in patients with Whipple’s disease. The response in uninfected individuals increased after restimulation and was characterized by the expression of CD154, indicating the specificity of the reaction (26). Proteinase K digestion of the T. whipplei lysate abolished stimulative capacity, pointing at proteins as stimulus. Cross-reactivity with apathogenic actinomycetes was not observed as the frequency of T. whipplei-specific Th1 cells (percent of IFN-{gamma}+ cells of CD4+ T cells) of control subjects was much higher and the reactivity to C. cellulans and C. hominis was similar in control subjects and in Whipple’s disease patients. In addition, neither endotoxin nor possible host factors from infected MRC5 were responsible for the T. whipplei-specific reactivity.

In healthy subjects, T. whipplei-specific peripheral Th1 cells were found in a frequency similarly as described for other Ags (27). This consistent T. whipplei-specific cellular immunity in healthy individuals is probably induced by regular contact of the enteric immune system with T. whipplei. Indeed, the presence of T. whipplei-specific IgG Abs in the serum and T. whipplei DNA in the gastrointestinal tract of some healthy individuals (7, 9, 10) support this hypothesis. Additionally, according to our results, active triathletes, exposed to open watercourses, revealed the highest percentages of T. whipplei-specific CD4+ T cells followed by the age matched, and finally the young subjects. TB patients showed reduced T. whipplei-specific responses compared with healthy subjects. Therefore, attributes that predispose for mycobacterial infection or the infection per se seem to impair reactivity to T. whipplei too.

In addition, the mucosal T cell response to T. whipplei was studied. In accordance with the results from the peripheral blood, T. whipplei-specific Th1 cells in duodenal lymphocytes of healthy subjects (cured gastric ulcer patients) were more frequent than in patients with Whipple’s disease. The activation status of the freshly isolated duodenal lymphocytes of these control subjects and the reactivity to SEB was significantly lower than in Whipple’s disease patients. Though, we cannot exclude definitively that former gastric ulcer disease influences Ag-specific reactivity to T. whipplei in the duodenum. But because we see no signs of general enhanced activation in the duodenal lymphocytes of those control subjects, we thus assume that the T. whipplei-specific reactivity is not enhanced by previous gastric ulceration. The reduced response of patients with Whipple’s disease to T. whipplei was consistent, and independent of disease activity and treatment. After T. whipplei-specific stimulation, the frequency of CD4+ cells expressing CD69, an early marker of activation (19), and CD154, a marker for the assessment of Ag-specific T cell responses (26), was reduced in Whipple’s disease patients compared with control subjects. Thus, insufficient T cell activation, and not only repressed IFN-{gamma} expression, accounts for reduced Th1 reactivity.

Impaired Th1 reactivity of PBMC (secretion of IFN-{gamma}) and proliferation to various mitogens in vitro has been described in Whipple’s disease (11, 12, 13, 14, 15). Reduced serum IgG2 (15), the association of T. whipplei-specific IgM with Whipple’s disease (10), and eradication of the causative organism through adjunctive IFN-{gamma} therapy in one patient (28) indicate the pathogenetic relevance of poor Th1 responses in vivo. However, SEB that was used in this study as unspecific stimulus seems to have similar efficiency in PBMC of Whipple’s disease patients and healthy subjects, just as described before for anti-CD2 respective anti-CD3 and anti-CD28 (15). Moreover, stimulation with SEB resulted in higher reactivity in CD4+ duodenal lymphocytes of Whipple’s disease patients than of healthy subjects. In addition, Ag-specific responses to tetanus toxoid, CMV, tuberculin, G. lamblia, and actinomycetes were comparable in PBMC of the study groups. Thus, the Th1 unresponsiveness of Whipple’s disease patients seems to be limited to only selected stimuli. Consequently, we hypothesize that a T. whipplei-specific immune defect contributes to the very rare susceptibility to the ubiquitously present bacteria. However, we cannot exclude that the actinomycete itself plays a role in the induction of this defect.

This hypothesis is supported by two major aspects: 1) CD4 immunodeficiencies (for example AIDS) do not seem to correlate with a more frequent incidence of Whipple’s disease, and 2) patients with Whipple’s disease do not suffer more often from opportunistic infections known to be associated with impaired CD4+ T cell activity like toxoplasmosis, Pneumocystis carinii, or nontuberculosis mycobacterial infections (29, 30). Only giardiasis was found recently to be associated with Whipple’s disease (25). However, the similar G. lamblia-specific Th1 reactivity in healthy subjects and Whipple’s disease patients casts a common predisposing immune defect into doubt.

We found enhanced cell activation of peripheral and duodenal T cells of Whipple’s disease patients independent of disease status as described (13, 31). Ongoing infection might be simulated by remnants of T. whipplei that are persistent in affected tissues (3) even after successful treatment. Correspondingly, suppression of T cell reactivity often occurring during persistent infection (32) might be responsible for the impaired reactivity of Whipple’s disease patients to mitogens (11, 12, 13, 14, 15). Hence, some of the proposed mechanisms for T. whipplei-specific unresponsiveness can be excluded. Cytokines like IL-10 (33) and TGF-beta (34) have been shown to act suppressively. However, in this study, Whipple’s disease patients did not show enhanced percentage of IL-10-producing CD4+ PBMC after stimulation with SEB and serum levels of TGF-beta have been shown to be similar compared with healthy subjects (16). Consequently, neither TGF-beta nor IL-10 seem to be suppressive factors in Whipple’s disease. Exogenous IL-2 did not result in an increase in T. whipplei-specific Th1 reactivity of Whipple’s disease patients. In addition, IL-2 induced enhanced expression of IFN-{gamma} only in preactivated T cells expressing CD25 of healthy subjects (35). Thus, T cell anergy or activity of regulatory T cells does not seem to explain the specific unresponsiveness we observed in Whipple’s disease.

Macrophages seem to be of central importance in the development of the disease (36). Recently, it has been shown that intestinal macrophages of Whipple’s disease patients display in vivo the phenotype of M2/alternatively activated macrophages (17) that favor the development of Th2 responses and inhibit protective Th1 polarization (37). This fact explains the deregulated Th1/Th2 response in Whipple’s disease we have previously described (14). In addition, untreated Whipple’s disease patients reveal elevated serum levels of IL-16 which favors T. whipplei replication in vitro (38). One hypothesis about the role of IL-16 in vivo is that it contributes to a general recruitment of CD4+ T cells in an inflammatory process resulting in cells responsive to cytokine stimulation, but refractory to Ag-specific stimulation (39) because it interferes with cell-mediated immune response (40) and induces tolerogenic dendritic cells (41). However, IL-16 alone was not sufficient to account for T. whipplei replication (38). Indeed, recent own studies refer to diminished monocyte functions and displacements of circulating dendritic cell phenotypes in Whipple’s disease (V. Moos, D. Kunkel, R. Ignatuis, M. Zeitz, and T. Schneider, manuscript in preparation). Thus, a general Th1-suppressive and tolerogenic intestinal milieu may lead to reduced degradation of invading T. whipplei, and disturbed processing and presentation of T. whipplei Ags. Hereditary genetic predisposition (42) and specific host factors (43) have been shown to influence the spread of intracellular bacteria and the deletion of a single IFN-{gamma}-inducible gene induced exclusive susceptibility to Toxoplasma gondii in mice (44). In the case of infection with T. whipplei, the invading pathogen might induce specific susceptibility in the Th1 system of predisposed human hosts that facilitates bacterial invasion and impairs immunological clearance. Although we cannot predict the detailed mechanism yet, we established a basis for further investigations with this study.

In summary, we have detected a reduced T. whipplei-specific Th1 response of Whipple’s disease patients while peripheral and mucosal T cells of healthy subjects and TB patients reveal a significant reactivity. A proposed T. whipplei-specific defect might explain why the ubiquitous bacillus causes symptomatic infection only in certain hosts.


   Acknowledgments
 
We thank Nicole Gatzemeier, Nadine Scholz-Neumann, and Jutta Immlau for technical assistance, and Christopher Previti for proofreading the manuscript. We are grateful to all patients and control subjects who took part in this study and thank the staff of the referring medical institutions for help in obtaining samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by European Union Contract No. QLG1-CT-2002-01049, Deutsche Forschungs Gemeinschaft KFO 104. Back

2 Address correspondence and reprint requests to Dr. Verena Moos, Charité, Campus Benjamin Franklin, Medizinische Klinik I, Hindenburgdamm 30, 12203 Berlin, Germany. E-mail address: verena.moos{at}charite.de Back

3 Abbreviations used in this paper: TB, tuberculosis; SEB, Staphylococcus enterotoxin B. Back

Received for publication February 14, 2006. Accepted for publication May 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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