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Clinical and Diagnostic Laboratory Immunology, January 2003, p. 59-65, Vol. 10, No. 1
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.1.59-65.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Immunological Memory Responses to Listeria monocytogenes in Rodents, as Measured by Delayed-Type Hypersensitivity (DTH), Adoptive Transfer of DTH, and Protective Immunity, following Lactobacillus casei Shirota Ingestion

R. de Waard,1* E. Claassen,1 G. C. A. M. Bokken,1 B. Buiting,2 J. Garssen,2 and J. G. Vos2,3

Department of the Science of Food of Animal Origin,1 Department of Veterinary Pathology, Utrecht University, Utrecht,3 Laboratory for Pathology and Immunobiology, National Institute of Public Health and the Environment, Bilthoven, The Netherlands2

Received 15 May 2002/ Returned for modification 22 July 2002/ Accepted 1 November 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effect of orally administered Lactobacillus casei Shirota (L. casei) on immunological memory, as measured by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR). The studies were performed in animal models in which the animals were rendered immune by a primary Listeria monocytogenes infection. It was shown that orally administered viable L. casei, and not heat-killed L. casei, enhanced significantly the antigen-specific DTH at 24 and 48 h in Wistar rats, Brown Norway rats, and BALB/c mice in a time- and dose-dependent fashion. L. casei had to be administered at least 3 days prior to the DTH assay at a daily dose of 109 CFU in order to induce significant effects. Long-term administration of 109 CFU of viable L. casei resulted in enhanced ACR, as demonstrated by reduced L. monocytogenes counts in the spleen and liver and diminished serum alanine aminotransferase activity after reinfection. Enhancement of cell-mediated immunological immune responses by L. casei was further established in an adoptive transfer study. Naïve recipient BALB/c mice, which were infused with nonadherent, immunized spleen cells from L. casei-fed donor BALB/c mice, showed significantly enhanced DTH responses at 24 and 48 h compared to recipient mice which received spleen cells from control donor mice. In conclusion, orally administered L. casei enhanced cell-mediated immunological memory responses. The effects relied on lactobacillus dose and viability as well as timing of supplementation and, further, appeared to be independent of host species or genetic background.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Various cells of the innate and acquired immune system interact in a concerted fashion to constitute a sophisticated set of responses to protect the physiological integrity of the host against infectious agents and tumors. Inappropriate orchestrated immune responses lead to, e.g., autoimmune and allergic disease states as well as enhanced susceptibility to infections. Different compounds are known which have the capacity to enhance or decrease biological responses of the immune system in order to restore disturbed immune responses or to elevate resistance to infectious agents. These compounds can be classified as so-called immunomodulators or biologic response modifiers (23).

Lactobacilli form a source of potential modulators of the immune system. It has been demonstrated that specific Lactobacillus strains can modulate host immunity, which positively correlates with enhanced resistance to various viral and bacterial infections (9, 20, 24). The immunomodulating effects are dependent on various factors, such as intrinsic adjuvanticity properties (21), dose (13, 31), viability (16, 20, 30), route and timing of administration of the specific Lactobacillus strains (i.e., oral versus parenteral), and the physiological state and genetic background of the host (20).

Previous results from our group established that 109 orally administered viable Lactobacillus casei Shirota strain YIT9029 (L. casei) bacteria given daily enhanced Trichinella spiralis antigen-specific immunoglobulin G2b (IgG2b) and delayed-type hypersensitivity (DTH) responses in Brown Norway and Wistar rats in an oral infection model of T. spiralis (8). The immunostimulating activity of Lactobacillus casei Shirota was also observed in a rat model of food-borne Listeria monocytogenes infection. It was shown in orally Listeria-infected rats that ingestion of L. casei enhanced the DTH reaction (7). From these studies, and based on the described immune cell populations involved in DTH responses (29), it was asserted that orally supplemented L. casei could exert a direct effect on T-helper-1-cell immunity by modulating the amount and activity of antigen-specific T lymphocytes and/or could exert an indirect effect on T-lymphocyte activity through stimulation of other cell types, such as phagocytes.

Collectively our studies prompted further examination of the effect of orally administered Lactobacillus casei Shirota strain YIT9029 on cell-mediated immunity in an animal model of systemic L. monocytogenes infection. Rats and mice, which are sublethally infected with viable L. monocytogenes, generate DTH and acquired cellular resistance (ACR) during the course of the infection (29, 36). ACR provides long-lasting, protective immunity to reinfection and can be measured as the pathogenic load after reinfection. Although the correlation between DTH and ACR remains controversial, they are both inversely related to the immune status of the host. Measurement of DTH and ACR forms a reliable method for examining the effects of orally administered lactobacilli on immunological memory.

In a parenteral Listeria monocytogenes infection model we studied the effects of orally administered Lactobacillus casei Shirota strain YIT9029 on T-cell-dependent immune responses by measurement of Listeria-specific DTH reaction and ACR in rechallenge and adoptive transfer studies. Since immune-modulating effects may rely on a range of factors, we investigated whether the effects induced by L. casei ingestion were dependent on the lactobacillus dose, viability, and administration timing as well as host genetics. In all experiments with actively infected Wistar rats, Brown Norway rats, and BALB/c mice, it was shown that ingestion of viable L. casei enhanced immunological memory, as determined by DTH and ACR, in a dose-, viability-, and timing-dependent fashion. Since DTH, ACR, and transfer of DTH are T-cell-dependent phenomena, it is suggested that L. casei-induced immunomodulation could be attributed, at least partly, to T memory cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals. Male specified-pathogen-free Wistar (U:Wu) rats, Brown Norway (Rij:Hsd) rats, and BALB/c mice were obtained from the animal facility of the Utrecht University (Utrecht, The Netherlands), Harlan Netherlands B.V. (Horst, The Netherlands), and the National Institute of Public Health and the Environment (Bilthoven, The Netherlands), respectively. The rats and mice, 6 to 7 weeks of age, were randomly allocated and kept in filter-topped cages (two animals per cage). The animals were provided with autoclaved food pellets and drinking water ad libitum. The animals were housed in the barrier under standard conditions (50 to 60% humidity, 12-h dark-12-h light cycle). All animal studies were approved by an independent ethical committee of the Utrecht Faculty of Veterinary Medicine, as required by Dutch law.

Bacteria. Lactobacillus casei Shirota strain YIT9029 (L. casei) was obtained from the Yakult Central Institute for Microbiological Research (Tokyo, Japan). L. casei was grown overnight at 37°C in MRS broth (CM359; Oxoid, Haarlem, The Netherlands) and resuspended in sterile saline (0.9% NaCl) to a final concentration of 2 x 109, 2 x 108, or 2 x 107 CFU/ml, respectively. The number and viability of the lactobacilli were determined by aerobic culturing on MRS plates (Oxoid CM361). Heat-killed L. casei was obtained by heating a viable L. casei suspension of 2 x 109 CFU for 15 min at 65°C. The animals received 0.5 ml of saline (control) or bacterial suspensions in saline (experimental groups) per day by a feeding tube placed in the esophagus.

Listeria monocytogenes strain L242/73 type 4b was obtained from the National Institute of Public Health and the Environment (Bilthoven, The Netherlands). To prepare activated L. monocytogenes for oral infections, an egg passage was performed as described by Ruitenberg et al. (32). The stimulated bacteria were maintained in brain heart infusion BHI broth (Oxoid CM225) at -80°C. In each experiment, an aliquot was thawed and clear-cultured on BHI (Oxoid CM375) agar plates. Inocula of L. monocytogenes were grown for 18 h in BHI broth and resuspended in sterile saline to the appropriate concentrations. The L. monocytogenes concentration was confirmed by aerobic culturing on both BHI and modified Oxford agar (MOA) plates. The MOA medium was modified from a selective medium for L. monocytogenes as described elsewhere (5).

Experimental design. In various independent experiments, the effect of orally administered Lactobacillus casei Shirota strain YIT9029 on L. monocytogenes-specific DTH responses and ACR in mice and rats was studied. The studies were designed to study whether the immune effects induced by oral L. casei intake are dependent on lactobacillus dose, viability, and administration timing and host genetics. In all experiments, the moment of L. monocytogenes infection was defined as time zero (t = 0).

Host genetics. In three separate experiments, the effect of L. casei on the DTH response in Wistar rats, Brown Norway rats, and BALB/c mice was studied. Starting 10 days (t = -10) before the subcutaneous L. monocytogenes infection, the animals (n = 6 per group) were daily administered in 0.5-ml suspensions, which contained 109 CFU of viable L. casei. Control animals received saline instead of lactobacilli. At day 10 postinfection (t = 10), the L. monocytogenes-specific DTH was measured.

Administration timing. Starting 10 days before (t = -10) or 7 days after (t = 7) the subcutaneous L. monocytogenes infection, rats were daily administered 0.5-ml suspensions with 109 CFU of viable L. casei. Control animals received saline instead of lactobacilli (n = 6 rats per group). At t = 7, the host has completely cleared the bacterial pathogen (data not shown), which means that the control and experimental groups have been exposed to the same amounts of Listeria antigen, since the animals were not L. casei treated before day 7 postinfection. Differences found in DTH responses at day 10 would be due only to ingestion of L. casei from day 7 till day 10 postinfection. At day 10 post infection (t = 10), L. monocytogenes-specific cell-mediated immunity was measured using the DTH assay.

Lactobacillus dose, viability, and administration timing. Starting 7 (t = 7), 8 (t = 8), or 9 (t = 9) days after the subcutaneous L. monocytogenes infection, rats were daily administered 0.5-ml suspensions containing 109 CFU of viable L. casei. Additionally, the effects of orally administered 109 heat-killed, 108, and 107 CFU of viable L. casei on the DTH response were analyzed. Control animals received saline instead of lactic acid bacteria (n = 4 rats per group). At day 10 postinfection (t = 10), L. monocytogenes-specific cell-mediated immunity was measured using the DTH assay. Subsequently, rats received daily the corresponding L. casei suspensions for an additional 8 weeks. At day 63 (t = 63) the animals were reinfected with L. monocytogenes, and 36 h later the rats were killed and analyzed for bacterial burden in the liver and the spleen as well as serum alanine aminotransferase (ALT) activity. ACR was determined by the capacity of the rats to eliminate L. monocytogenes cells from the spleen and liver after the challenge.

Primary and challenge infections. Rats and mice were subcutaneously infected at day zero with 5.105 or 2.104, respectively, viable L. monocytogenes cells at five different sites, 0.1 ml at two sites in the left and right thighs, 0.1 ml at two sites in the left and right forelimbs, and 0.1 ml in the neck region. Challenge infections were performed by intraperitoneal infusion of 5.107 viable L. monocytogenes cells in both rats and mice.

Adoptive transfer of immunity. Starting 10 days previously (t = -10), subcutaneously L. monocytogenes-infected donor mice received orally 109 CFU of L. casei or saline per day (n = 10 mice per group). Seven days (t = 7) after the primary L. monocytogenes infection, single-cell suspensions were prepared from the spleens of donor mice. After passage over a column packed with glass wool fiber, 108 wool-nonadherent spleen cells of five mice of each group were infused via the orbital plexus into syngeneic naive recipients. The recipient mice received no lactobacilli. Twenty-four hours after the transfer (t = 8), the DTH was measured in the recipient mice and the remaining five donor mice of each group. Twenty-four and forty-eight hours after the challenge, the increase in ear thickness was measured. Five days after setting the DTH assay (t = 13), all mice were reinfected with live L. monocytogenes. The mice were killed 24 h later and analyzed for L. monocytogenes counts in the liver and spleen. ACR was determined by the capacity to eliminate L. monocytogenes cells after challenge.

DTH assay. Animals in the experimental and control groups were challenged in left and right ear pinnas with 25 µl (107 CFU) (for rats) 10 µl (107 CFU) of heat-killed L. monocytogenes (HKLM) under light ether anesthesia. The application and preparation of HKLM in DTH assays have been described in previous studies (1, 12, 14). The absolute increase in ear thickness was measured with a digital engineer's micrometer (Mitutoyo, Veenendaal, The Netherlands) 24 and 48 h after challenge. The swelling was calculated according to the following equation: net swelling = (T24 - T0) - (P24 - P0); T0 is thickness of ear before HKLM challenge, T24 is thickness of ears 24 h after HKLM antigen challenge, P0 is thickness of ear before phosphate-buffered saline injection, and P24 is thickness of ear 24 h after phosphate-buffered saline injection. The same type of equation for DTH measurements at 48 h after challenge was performed. It was shown that challenging the ear pinnas in noninfected L. casei-fed rats with heat-killed L. monocytogenes or challenging the ear pinnas of L. monocytogenes-immunized rats with heat-killed L. casei did not result in a significant net swelling response. This excludes possible cross-reactivity of L. monocytogenes antigens with L. casei antigens.

Bacteriological analysis. Spleens and livers were removed aseptically from the animals. The numbers of viable L. monocytogenes were determined by culturing 100 µl of serial 10-fold dilutions of organ homogenates in saline on MOA plates. The numbers of L. monocytogenes bacteria were expressed as CFU per gram of spleen and/or liver. Detection limit was 100 CFU/g of tissue.

ALT. ALT levels in rate serum were analyzed with an automated chemistry analyzer (Beckman Synchron CX7; Beckman-Coulter Ned., Mijdrecht, The Netherlands). Alanine aminotransferase is a liver-specific enzyme in rats (3). Analyses were performed at the Department of Clinical Sciences of Companion Animals of the Utrecht University, The Netherlands.

Statistics. Statistical analysis was performed with SPPS software (SPPS version 7.5.2.). Significant differences were revealed by analysis of variance followed by a Student's t test for comparison between control and experimental groups. A P value of <0.05 is considered significantly different. A low number (100 CFU) was assigned to animals from whom no L. monocytogenes could be recovered in order to calculate a group mean. To analyze correlations between (i) dose of L. casei and DTH responses, (ii) timing of L. casei administration and DTH responses, and (iii) L. monocytogenes counts in liver and ALT levels in serum, the Pierson correlation test was applied. Values are expressed as means ± standard deviations (SD).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Host genetics and the effects of oral L. casei administration on DTH. Figure 1 shows that L. casei, following oral delivery, significantly enhanced the L. monocytogenes-specific DTH response in Wistar rats at 24 h (P < 0.05) and 48 h (P < 0.05), compared to results for control animals. In a parallel study it was shown that orally administered L. casei enhanced significantly the expression of DTH at 24 h (P < 0.05) and 48 h (P < 0.05) in the Brown Norway rats (Fig. 1). Further, in two experiments with L. monocytogenes-immunized BALB/c mice, it was demonstrated that L. casei ingestion enhanced the antigen-specific DTH in rats. In the first experiment the DTH was significantly enhanced at 24 h (P < 0.01) and 48 h (P < 0.01) after challenging the ear pinna (Fig. 1). In the second experiment DTH values in lactobacillus-treated mice were significantly higher at 24 h (P < 0.001) and 48 h (P < 0.05) (Table 1). In these experiments a dose of 109 CFU of viable L. casei was orally administrated on a daily basis, starting 10 days prior to the subcutaneous L. monocytogenes infection.



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  		FIG. 1. Effect of L. casei Shirota on L. monocytogenes-specific DTH response in Wistar rats, Brown Norway (BN) rats, and BALB/c mice. Daily oral administration of L. casei started 10 days prior to the L. monocytogenes infection. Following 10 days after the subcutaneous L. monocytogenes infection, DTH responses were measured as the difference in ear thickness (in no. of millimeters x 10-2) before and 24 to 48 h after intradermal challenge. Bars represent the means ± standard deviations for six animals per group. Values are significantly different from those for control animals: *, P < 0.05; **, P < 0.01, as calculated with Student's t test.

 

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  		TABLE 1. DTH and adoptive transfer of ACR with splenocytes from L. casei Shirota-fed donor mice infected subcutaneously with viable L. monocytogenes

 
The effect of L. casei administration timing on DTH. Starting daily, oral L. casei administration at a dose of 109 CFU, 10 days prior to as well as 7 days after the primary Listeria monocytogenes infection in Wistar rats, resulted in significantly enhanced antigen-specific DTH expression, as shown in Fig. 2. The magnitude of the DTH responses at 24 and 48 h was comparable for the two groups of rats which received L. casei 20 or 3 consecutive days prior to the DTH assay.



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  		FIG. 2. Effect of L. casei Shirota on L. monocytogenes-specific DTH response at 24 and 48 h after challenge in Wistar rats. Daily oral administration of L. casei started 10 days before [L. casei (t = -10)] and 7 days after [L. casei (t = 7)] infection with L. monocytogenes. Following 10 days after (t = 10) the subcutaneous L. monocytogenes infection, DTH responses were measured. Bars represent the means ± standard deviations for six animals per group. Values are significantly different from those for control animals: **, P < 0.01, as calculated with Student's t test.

 
The effect of oral administration of 109 CFU of viable L. casei 3 days prior to the DTH assay in Wistar rats was reproduced, as shown in Fig. 3. A significantly enhanced Listeria-specific DTH reaction was measured at 24 h (P < 0.05) and 48 h (P < 0.01) in animals which received L. casei starting 3 days before the DTH assay. DTH values at 24 and 48 h after ingestion of 109 CFU of lactobacilli at 2 days or 1 day prior to the DTH assay did not significantly differ from those for control rats.



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  		FIG. 3. Effect of differentially dosed, timed, viable, and dead L. casei Shirota on L. monocytogenes-specific DTH responses at 24 and 48 h after challenge in Wistar rats. Rats received a daily oral dose of saline (i), 109 CFU of heat-killed L. casei (ii), or 107 (iii), 108 (iii), or 109 CFU of viable L. casei (ii, iii, and iv) starting 7 days after the L. monocytogenes infection (t = 7) or 109 CFU of viable L. casei starting 8 (t = 8; iv) or 9 days (t = 9; iv) after the L. monocytogenes infection. Following 10 days (t = 10) after the subcutaneous L. monocytogenes infection, DTH responses were measured. Bars represent the means ± standard deviations for four animals per group. Values are significantly different from those for control animals: *, P < 0.05; **, P < 0.01, as calculated with Student's t test. #, identical data bars.

 
Decreasing the number of lactobacillus applications prior to the DTH assay resulted in a strong, but not significant, tendency to correlate with decreasing DTH values at 24 h. This correlation was significant at 48 h (r = 0.55; P < 0.05).

The effect of L. casei viability on DTH. Ingestion of heat-killed L. casei did not affect DTH expression. As shown in Fig. 3, DTH values at 24 and 48 h were not significantly different from control values after oral administration of 109 CFU of heat-killed L. casei 3 days prior to the DTH assay.

Effect of L. casei dose on DTH. DTH values at 24 and 48 h were not significantly different from control values after oral administration of 108 and 107 viable L. casei bacteria 3 days prior to the DTH assay, as denoted in Fig. 3. A significant correlation was observed between lactobacillus dose, administered 3 days prior to the DTH assay, and DTH values, measured at 24 and 48 h after challenge (r = 0.61 and P < 0.05 [24 h]; and r = 0.71 and P < 0.01 [48 h]).

Effects of long-term oral administration of viable L. casei on ACR. Long-term (i.e., approximately 8 weeks) oral supplementation of viable L. casei at a dose of 109 CFU, but not 107 or 108 CFU, enhanced ACR as measured by reduced counts of viable L. monocytogenes in the spleen and liver at 36 h after reinfection compared to results for control animals (Table 2). Immunologically primed animals responded to reinfection in a more vigorous and effective manner, as shown in Table 2. Unprimed control animals (control-1) had approximately 10 times higher Listeria counts in the visceral organs, spleen (P < 0.01), and liver (P < 0.01), as well as significantly higher serum ALT activity (P < 0.01) than primed control animals (control-2).


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  		TABLE 2. Effect of L. casei Shirota dose, viability, and timing on ACR and ALT concentrations in serum in Wistar rats after reinfection

 
Further, L. monocytogenes growth was not affected by long-term oral administration of heat-killed L. casei. Serum ALT activity was significantly lower in two (out of three) groups of animals which were fed viable L. casei Shirota at a dose of 109 CFU per day for 63 consecutive days, compared to the control group (control-2), as shown in Table 2. Other experimental groups did not affect significantly the ALT activity. In spite of that, ALT values were significantly correlated with bacterial load in the liver (r = 0.75; P < 0.01).

Adoptive transfer of immunity. It was shown that expression of antigen-specific DTH was significantly enhanced in recipient BALB/c mice which received nonadherent spleen cells from L. casei-fed donor BALB/c mice. In Fig. 4, it is shown that DTH was enhanced at 24 h (P < 0.01) and 48 h (P < 0.05). The magnitude of the DTH values of recipient mice, as depicted in Fig. 4, was comparable to DTH values of the remaining, corresponding groups of mice (Table 1) that were assayed parallel to the spleen cell-infused recipient mice, following 7 days after the primary infection with L. monocytogenes. Ten days after the DTH assay, all groups of mice were reinfected with viable L. monocytogenes. Numbers of Listeria bacteria in the spleen appeared reduced, but not significantly, in normal L. casei-fed mice as well as in reconstituted recipients which received spleen cells from L. casei-supplemented donor animals, compared to the respective controls.



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  		FIG. 4. Transfer of L. monocytogenes-specific DTH. BALB/c donor mice were subcutaneously infected with viable L. monocytogenes. Starting 10 days prior to infection, the mice received a daily dose of L. casei or saline. Nonadherent spleen cells from L. casei and control-fed BALB/c mice were infused into syngeneic naive recipients 7 days after infection. Twenty-four hours after the transfer, the L. monocytogenes-specific DTH was set, and it was determined 24 and 48 h later for the recipient mice. Bars represent the means ± standard deviations for five animals per group. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from the control group as calculated with Student's t test.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The immune system can be optimized through oral supplementation of specific Lactobacillus strains (2, 9). Modulation of B-cell-mediated immune responses can be monitored relatively simply by, for instance, measurement of titers of antigen-specific antibody in serum. In vivo measures of T-cell-mediated immunity comprise delayed-type hypersensitivity responses and reinfections with intracellular pathogens, such as L. monocytogenes (11, 36).

In the present study it is shown that ingestion of a daily dose of 109 CFU of live, L. casei Shirota strain YIT9029, starting at least 3 days prior to setting the L. monocytogenes-specific DTH assay, significantly enhances the DTH response at 24 and 48 h in the genetically different Wistar and Brown Norway rats as well as in BALB/c mice (Fig. 1, 2, and 3). Brown Norway rats and BALB/c mice are representative for animals which tend to respond immunologically in a more Th2-skewed fashion than Wistar rats (17, 28, 38, 40). Our observations demonstrated that the effect of L. casei is not dependent on host genetics. Host-independent effects by L. casei have also been shown in previous other studies of our group. With T. spiralis-infected Wistar and Brown Norway rats it was shown that oral L. casei supplementation enhanced both the antigen-specific DTH and serum IgG2b. In our hands, enhancement of antigen-specific DTH by peroral L. casei application has also been observed in a food-borne model of L. monocytogenes (7). Expression of antigen-specific DTH in rats and mice and of IgG2b responses in rats are reported to be manifestations of Th1-type cell-mediated responses (15, 33). The capacity to stimulate Th1 activity by L. casei has also been suggested in previous in vivo and in vitro studies with mice performed by other groups (18, 25, 37). From these results it was suggested that oral administration of L. casei leads to a prominent state of cellular innate immunity via phagocyte activation, which subsequently enhances Th1 cell activity. The results from this and other laboratories strongly suggest that L. casei has a genuine effect on Th1-cell-mediated immunological memory.

Adoptive transfer studies with mice increasingly confirmed the involvement of T memory cells in the enhanced DTH response after L. casei application. Ample evidence exists that DTH to heat-killed L. monocytogenes or soluble Listeria antigens is transferred by spleen-derived CD4+ T-helper cells (1, 35, 39). The in vivo effector assay comprised the transfer of nonadherent spleen cells from L. monocytogenes-infected donor BALB/c mice, which were fed L. casei or control saline, to naive recipients, which were challenged 24 h later in the ear pinna. The results demonstrated that naive recipient mice, which received nonadherent spleen cells from L. casei-fed donor mice, had enhanced DTH responses compared to control animals. This finding supports our view that L. casei ingestion enhances the activity and/or proliferation of T- helper-1 memory cells.

Although the association of DTH and acquired cellular immunity remains controversial, they both are manifestations of T-cell-mediated immunological memory (1, 6, 27, 35, 39). The kinetics of development and endurance of both responses have been reported to be similar (22). Presumably, different subsets of T cells are involved in DTH and ACR (1, 26, 39). Despite the uncertain association between DTH and ACR, it was shown that long-term, i.e., for approximately 8 weeks, oral administration of a daily dose of 109 CFU of L. casei enhanced acquired cellular resistance against a reinfection with L. monocytogenes as measured by reduced numbers of bacterial pathogen in the spleen and liver as well as reduced ALT levels in serum, next to the enhancement of DTH. In the transfer studies with mice, the bacterial counts in the spleen also appeared to be decreased after reinfection, albeit this was statistically not significant.

Further, it was shown that the effects of L. casei on immunological memory responses were dose dependent. Incrementally increasing doses of L. casei significantly conveyed higher levels of enhancement of DTH and ACR. A dose of 107 L. casei bacteria was shown to be ineffective in modulating DTH or ACR. It is ambiguous whether the effective dose of L. casei is associated with a disparity in sensitivity of the specific cells involved in the measured immune parameters. It has been shown that a specific Lactobacillus rhamnosus strain was able to enhance the phagocytic capacity of white blood cells at a dose of 107, while a 100-times-higher dose of lactobacilli was needed to increase the phagocytic capacity of peritoneal cell preparations, following oral delivery (13).

In our specific experimental setup with L. monocytogenes-infected rats, it is possible that viable and killed lactobacilli exert different effects, for example, through differential stimulation of dendritic cell subsets (DC1 versus DC2), which control the directional development of T-helper-cell phenotypes (4, 19). Another explanation for differential effects of viable and dead L. casei, as shown in our studies, could be that immune stimulation by L. casei is mediated by heat-sensitive structures on the bacterial surface. Data obtained by Haller et al. showed that higher concentrations of heat-killed lactobacilli were needed to induce the same tumor necrosis factor alpha production by monocytes as was induced by live lactobacilli (16). It was speculated from these studies that heat-killed lactobacilli express less undisturbed surface modulins (i.e., peptidoglycans or lipoteichoic acids) which can be recognized by pattern recognition receptors (e.g., CD14 or toll-like receptors) on various antigen-presenting cells (10, 34).

In summary, the present study endorses the idea that orally administered L. casei Shirota strain YIT9029 enhances cell-mediated immunological memory responses, as determined by DTH, ACR, and adoptive transfer of DTH in an L. monocytogenes infection model. Since DTH, ACR, and transfer of DTH are T-cell-dependent phenomena, it is suggested L. casei-induced immunomodulation is mediated, at least partly, by memory T cells. Still, the nature of the cells, in terms of cytokine profiles and cell surface markers, in the observed effects remains to be established. Nevertheless, the effect of L. casei on cell-mediated memory responses relied on the timing of administration, was dose dependent, and possibly was imparted through a heat-sensitive constituent of L. casei. The effects of viable L. casei appeared to be independent of the responding host species and genetic background.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of the Science of Food of Animal Origin, Utrecht University, Yalelaan 2, 3508 TD Utrecht, The Netherlands. Phone: 31-30-2743485. Fax: 31-30-2744437. E-mail: dewaard{at}hi.nl. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Clinical and Diagnostic Laboratory Immunology, January 2003, p. 59-65, Vol. 10, No. 1
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.1.59-65.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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