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Articles

Kefir Milk Enhances Intestinal Immunity in Young but Not Old Rats¹

Karine Thoreux^*,,**2 and Douglas Lees Schmucker^*,

^* Cell Biology and Aging Section (151E), Department of Veterans Affairs Medical Center, San Francisco, CA 94121; Department of Anatomy, University of California, San Francisco, CA; and ^** Centre International de Recherche Daniel Carasso, 92350, Le Plessis-Robinson, France.

²To whom correspondence should be addressed. E-mail: coach{at}itsa.ucsf.edu.

	ABSTRACT

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The adjuvant effect of kefir fermented milk on the mucosal and systemic immune systems was examined in young (6 mo old) and old (26 mo old) rats. Kefir-fed rats consisted of young or old rats consuming kefir-fermented milk ad libitum on a daily basis in addition to the standard diet, for 28 d. Control rats consumed only the standard diet. The rats were immunized intraduodenally with cholera toxin (CT) on d 7 and 21 and killed on d 28. The nonspecific serum immunoglobulin (Ig)A titers in kefir-fed and control rats did not differ in either age group. The serum anti-CT IgA antibody concentrations were significantly higher in the kefir-fed young rats compared with their age-matched controls (+86%, P 0.05). This difference was associated with enhanced in vitro antibody secretion by cultured lymphocytes isolated from the Peyer’s patches and the intestinal lamina propria (+180%, P 0.05). These enhanced responses were found only in the young rats. However, the nonspecific serum IgG titer was higher (>120%, P 0.05) and the anti-CT IgG titer was lower (-80%, P 0.05), in both young and old kefir-fed rats compared with their respective controls. Nevertheless, these results demonstrate that a kefir-supplemented diet affects the intestinal mucosal and systemic immune responses to intraduodenal CT differently in young and old rats. Most importantly, our data suggest that orally administered kefir enhances the specific intestinal mucosal immune response against CT in young adult, but not in senescent rats.

KEY WORDS: • kefir • intestinal immunity • cholera toxin • aging • rats

	INTRODUCTION

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A number of studies have noted the immunomodulatory properties of probiotic organisms, especially the lactobacilli. Lactic acid bacteria (LAB)³and other probiotic organisms in fermented milks appear to be beneficial in the treatment of certain diarrheas, as well as in the stimulation of immune function (Sanders 1993 ). Consumption of yogurt or milks fermented with Lactobacillus casei, L. acidophilus, Bifidobacterium longum and mixtures of several LAB, as well as bacterial cell lysates, increases indices of immune response, e.g., the numbers of immunoglobulin (Ig)A-producing cells and macrophages, (Ig)G titers, and specific antibody responses to antigenic challenges compared with animals not consuming LAB (Isolauri et al. 1993 , Perdigón et al. 1993 and 1994 , Portier et al. 1993 , Popova et al. 1993 , Takahashi et al. 1998 , Tejada-Simon et al. 1999 ). Milk fermented with L. casei or with L. acidophilus and bifidobacteria improved the humoral immune responses in humans after challenges with rotavirus vaccine and an attenuated Salmonella typhimurium (Ty21a), respectively, (Isolauri et al. 1995 , Link-Amster et al. 1994 ).

Kefir is a stirred beverage made from milk fermented with a complex mixture of bacteria, including various species of lactobacilli, lactococci, leuconostocs, and aceterobacteria and yeasts (both lactose-fermenting and nonlactose-fermenting). Kefir differs from yogurt and other fermented milks in that kefir grains (small clusters of microorganisms held together in a polysaccharide matrix) or mother cultures from grains are added to milk and cause fermentation (Hallé et al. 1994 ). Despite the lack of data, recent studies suggest antibacterial, immunologic and antitumor effects of kefir in animals (Furukawa et al. 1990 and 1991 , Zacconi et al. 1995 ). Kefir and sphingomyelin isolated from the lipids in kefir have been reported to stimulate the immune system in both in vitro and in vivo studies (Furukawa et al. 1991 , Osada et al. 1994 ). Kefir also exhibits antimicrobial activity in vitro against a wide variety of gram-positive and gram-negative bacteria and some fungi (Cevikbas et al. 1994 , Zacconi et al. 1995 ).

Aging compromises the intestinal mucosal immune response in animals and humans [see Schmucker and Owen (1997) for a review]. Consequences of this immune dysfunction include a decline in the efficacy of mucosal vaccines, as well as increases in the incidences of infectious diseases and in the associated rates of morbidity and mortality in the elderly (Jeandel et al. 1996 , Owen and Lew 1995 , Schmucker et al. 1985 ). Although serum IgA levels increase in the elderly, the mucosal responses to antigenic challenges decline in rodents, primates and humans as a function of increasing age (Arranz et al. 1992 , Ebersole et al. 1985 , Paganelli et al. 1994 , Smith et al. 1983 , Taylor et al. 1992 ). Significant declines in the intestinal IgA antibody responses to intraduodenal cholera holotoxin (CT) have been demonstrated in rats (Schmucker et al. 1988) and rhesus macaques (Taylor et al. 1992 ). Although the perception persists that eating functional foods has beneficial effects on health maintenance during aging, there are few studies in which the effects of probiotic consumption have been examined. Therefore, this study was designed to assess the immumodulatory effect of kefir on the intestinal mucosal immune response to cholera toxin in young adult and senescent rats.

Lactobacilli may stimulate intestinal mucosal immunity in elderly humans (De Simone et al. 1992 and 1993 , Van de Water et al. 1999 ). De Simone et al. (1992) noted an increase in the B-cell concentration of peripheral blood and a decrease in colonic inflammatory infiltration in a group of elderly subjects (>70 y) after ingestion of Bifidobacterium bifidum and L. acidophilus. Long-term yogurt consumption increases interferon- production by adult human T lymphocytes and decreases allergic symptoms in elderly people (Halpern et al. 1991 , Van de Water et al. 1999 ). However, there is little information concerning the role of probiotics as adjuvants or the mechanisms whereby these organisms enhance mucosal immunity, including in the elderly.

	MATERIALS AND METHODS

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Kefir preparation

Kefir was prepared by the Centre International de Recherche Daniel Carasso, Le Plessis-Robinson, France. It consisted of ultrahigh temperature sterilized bovine milk with a fat content of < 2% (commercial source) fermented overnight by the addition of kefir grains. Each batch was made with the same starter kefir grains, using the same fermentation conditions to ensure that the probiotic constituents were similar, stored at 4°C and used within 3 wk. The proximate composition of the kefir corresponds to the milk composition used during the fermentation, i.e., 3.5 g/kg protein, 4 g/kg carbohydrate and 2 g/kg fat.

Animals

Male Fischer 344/NHsd rats from the National Institute on Aging colony (Harlan Sprague Dawley, Indianapolis, IN) were used throughout these studies. Rats, in good health and showing no signs of disease, were divided into young adult (6 mo old) and senescent (26 mo old) age groups. Rats aged 26 mo are beyond 50% survivorship and are considered senescent. The rats were housed individually under barrier conditions and consumed the standard NIH-31 open formula (18 g/kg protein, 4 g/kg fat, 5 g/kg fiber, 8 g/kg vitamin and minerals, NIH national stock number 8710–01-005–8438) and water ad libitum. All rats were maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources.

Experimental design

    Diets. Rats were acclimated for 1 wk before initiation of experiments. They were segregated into four groups of 5 rats each as follows: young kefir, old kefir, young control and old control. The young and old kefir groups received kefir in addition to drinking water each night for 7 d (d 1–7) before immunization and throughout the remainder of the study, i.e., until killed on d 28.

    Cholera toxin immunization. Rats were immunized intraduodenally with CT (100 µg, List Biological Laboratories, Campbell, CA) in 0.5 mL 0.01mol/L PBS (pH 7.4) containing 2g/L gelatin. A small midabdominal laparotomy was performed under isofluorane anesthesia (Marsam Pharmaceuticals, Cherry Hill, NJ), and CT was introduced directly into the duodenum immediately distal to the sphincter of Oddi using a tuberculin syringe with a 28-gauge needle (Schmucker et al. 1988 ). Rats were primed on d 7 and boosted on d 21. Seven days after boosting (d 28), groups of rats were food deprived overnight and killed by isofluorane anesthesia followed by bilateral thoracotomy.

Serum collection and preparation of cell suspensions

Blood samples were collected from the inferior vena cava while the rats were under anesthesia. After overnight incubation at 4°C and centrifugation at 1000 x g for 15 min, the serum was isolated, divided in aliquots and stored at -80°C until used. Single mononuclear cell suspensions were prepared from spleen (SP) and mesenteric lymph nodes (MLN) by teasing the tissue through sterile 100-µm cell strainers (Falcon, Becton Dickinson, Bedford, MA). The resulting single-cell suspensions were centrifuged at 2500 x g for 28 min over a density separation medium (Lympholyte-Rat, Cerdarlane Laboratories Limited, Toronto, Canada). The cells were washed 3 times and suspended in RPMI-1640 medium containing 10% fetal bovine serum, 25 mmol/L HEPES, 2 mmol/L glutamine, 1 x 10⁵ U/L penicillin, 100 mg/L streptomycin and 50 mg/L gentamicin (complete medium). Peyer’s patches (PP) were excised from the small intestine and cell suspensions were prepared by mechanical disruption using a syringe, a 19-gauge needle and a sterile cell strainer. Tissue fragments were removed by filtering the cell suspension through a glass wool column. Lamina propria (LP) lymphocytes were isolated from PP-free small intestinal segments previously flushed with calcium/magnesium-free Hank’s balanced salt solution as described previously (Lycke 1986 ). The tissue was dissociated by several consecutive incubations in complete medium containing 1 x 10⁴ U/L of collagenase (type VII, Sigma Chemical, St. Louis, MO). The resultant cell suspensions were diluted to final concentrations in complete medium, and cell viability was determined by trypan blue exclusion.

In vitro cell cultures

Suspensions of SP, MLN, PP and LP mononuclear cells (1 x 10⁴ and 1 x 10⁶ cells/well) were incubated in complete medium in 96-well round-bottom culture plates for 5 d at 37°C in a 5% CO₂ environment.

Detection of antibody-secreting cells by enzyme-linked immunospot (ELISPOT)

The numbers of IgA- and anti-CT IgA–secreting cells were determined by ELISPOT assay (Czerkinsky et al. 1983 ). Nitrocellulose plates (Millititer HA, Millipore, Bedford, MA) were coated overnight with either sheep anti-rat IgA (5 mg/L, The Binding Site, San Diego, CA) for IgA-secreting cells or monosialoganglioside-GM1 (Sigma) followed by CT (5 mg/L) for CT IgA–secreting cells. The plates were blocked with complete medium (3 h at 37°C) and inoculated with 100 µL of diluted cell suspensions. After overnight incubation (37°C), the cells were washed 10 times with PBS containing 0.05% Tween 20. IgA-secreting cells were detected by incubating in 100 µL of biotinylated goat anti-rat IgA (2 g/L at 1:2000 dilution, Rockland Immunochemicals, Gilbertsville, PA) in PBS-Tween 20 (2 h), followed by avidin-horseradish peroxidase (1:1000, Zymed Laboratories, South San Francisco, CA) for 1 h at room temperature. The ELISPOT was developed by adding 100 µL of 1.6 mmol/L 3-amino-9-ethylcarbazole in 0.1mol/L sodium acetate buffer containing 0.015% H₂O₂ (Kit AEC, Sigma) to each well. After the wells were flushed with water, the secreting cells were counted using a stereomicroscope; data were expressed as the mean ± SEM of IgA- or anti-CT IgA–secreting cells per 10⁶ cells.

Detection of antibodies by ELISA

Nonspecific IgA and IgG levels, as well as anti-CT IgA and anti-CT IgG antibody titers, in the culture media and sera were measured by ELISA. Microtiter wells were coated with CT (10 mg/L), sheep anti-rat IgA or IgG (2.5 mg/L, The Binding Site) and incubated sequentially with 20 g/L bovine serum albumin, 75 µL of serially diluted serum or culture supernate, biotinylated goat anti-rat IgA or IgG (1:5000, Rockland Immunochemicals), avidin-horseradish peroxidase (1:1000, Zymed Laboratories) and reacted with o-phenylenediamine (Zymed Laboratories). Rats fed the vehicle alone were used as negative controls. Values for nonspecific Ig concentrations and antibody titers were calculated from the linear portions of IgA or IgG standard curves and expressed as mg Ig/L of serum or ng Ig/10⁶ cells.

Statistical analysis

All results are expressed as the mean ± SEM. Statistical analyses were performed using two-way ANOVA followed by the Student-Newman-Keuls test (InStat software, version 2.01, GraphPad Software, San Diego, CA). Differences were considered significant when P 0.05.

	RESULTS

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Body weight and kefir consumption

Consumption of kefir was 41.8 ± 1.3 mL/d in the young and 46.9 ± 1.3 mL/d in the old rats (Table 1 ). When these data were normalized for body weight, the ratio was similar in the two age groups [10 mL/(100g · d), P > 0.05]. Although young rats gained 2% of initial body weight and old rats lost 10% of initial body weight over the 28-d experiment, these changes in weight were not significant over time. There was no significant difference in the pattern of body weight change between the control and kefir-fed rats within each age group (data not shown). No diarrhea, loss of appetite or discomfort was observed during the feeding trial.

Nonspecific total serum IgA titers did not differ between the control and kefir-fed rats within each age group (Table 2 ). The serum anti-CT IgA concentration was lower in old kefir-fed rats compared with the age-matched controls (-40%, P 0.01). This decline was reflected in the lower anti-CT IgA/nonspecific IgA ratio in the old group. On the contrary, feeding kefir to young rats enhanced the mucosal immune response as evidenced by a significantly higher serum anti-CT IgA titer compared with the respective controls (+86%, P 0.05). Interestingly, the nonspecific total serum IgG titers in both young and old kefir-fed groups were much greater compared with their respective controls (+125% and + 520%, respectively, P 0.01) (Table 2) . However, feeding kefir to young and old rats decreased the systemic IgG response to CT; the anti-CT IgG titers were significantly lower in both kefir-fed groups compared with their age-matched cohorts (-80%, P 0.01).

IgA secretion by cultured lymphocytes was not affected by diet because there were no differences between cells isolated from control and kefir-fed rats in either age group (Table 3 ). In old rats, the kefir-supplemented diet did not influence in vitro anti-CT IgA secretion. However, anti-CT IgA antibody secretion by intestinal LP cells isolated from kefir-fed old rats was 25% greater than that by similar cells isolated from control old rats (P = 0.55). In vitro antibody CT IgA secretion by PP and intestinal LP cells from young kefir-fed rats was 180% greater (P 0.05) than that measured in the control rats and secretions by the SP of kefir-fed rats were 50% (P 0.05) greater.

Although the differences in numbers of antibody-secreting cells were not significant in most cases, kefir-fed young rats yielded more anti-CT IgA antibody-secreting cells in the SP (+15%, P = 0.38), MLN (+42%, P = 0.096), PP (+21%, P = 0.24) and LP (+22%, P = 0.32) compared with their age-matched controls (Table 4 ). However, the number of anti-CT IgA secreting cells was lower in the SP (-32%, P = 0.19), MLN (-55%, P = 0.02), PP (-17%, P = 0.42) and LP (-23%, P = 0.23) of kefir-fed old rats compared with their controls.

	DISCUSSION

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Increased immunoglobulin secretion appears to be associated with a higher number of antibody-secreting cells in the gut-associated lymphoid tissues. Mice fed yogurt for up to 10 d exhibited a marked infiltration of plasma cells and lymphocytes into the intestinal mucosa (Perdigón et al. 1994 ). After 7 d, there was an increase in the number of IgA-secreting cells, although the numbers of IgG- and IgM-secreting cells, as well as T cells, were unaffected. Our results with kefir, together with data from others studying different fermented milks, suggest that these probiotic foods may have a common adjuvant effect on the mucosal immune system.

Our results suggest that kefir enhances the serum nonspecific total IgG titer in both young and old rats, whereas the nonspecific total IgA titer is similar in kefir-fed and control groups. On the contrary, the anti-CT IgA antibody titer is enhanced in kefir-fed young rats, whereas the IgG antibody titer is diminished. The serum anti-CT IgA titer most likely reflects circulating antibodies destined for mucosal sites. Our interpretation is supported by the observation that kefir increases anti-CT IgA secretion in cells isolated from the inductive and effector sites of the small intestine (PP and LP).

Cholera holotoxin stimulates both mucosal and systemic immunity and serves as a strong adjuvant, i.e., it induces an intestinal secretory IgA response to itself and to coadministered antigens, and it mediates an IgA memory response (Elson and Dertzbaugh 1999 , Snider 1995 ). The decline in the serum anti-CT IgG titer despite the increase in the nonspecific serum IgG titer in kefir-fed rats, likely reflects the adjuvant effect of CT, i.e., enhancing systemic immune responses against antigens present in the kefir. However, at the mucosal site, CT is recognized as a potent antigen and kefir appears to function as an adjuvant. Other investigators have reported that nonspecific serum IgG titers remain unchanged in animals consuming fermented milk or yogurt (Portier et al. 1993 , Tejada-Simon et al. 1999 ).

Although our results suggest that kefir does not enhance the intestinal mucosal immune response to CT in old rats, there are several inherent caveats in this study. First, the marked heterogeneity of responses within the old-age groups diminishes the number of significant differences detected within this age group, i.e., between kefir-fed and control rats. Second, we examined only two ages, young adult and old rats; as a result, we may have missed changes associated with maturation. Such studies should be performed across the entire life span of the animal model, rather than at the extremes. Third, the absence of rats fed nonfermented milk precludes our eliminating an effect attributable to milk alone. However, many other studies have demonstrated specific effects due to fermented milk compared with diets supplemented with nonfermented milk only (Furukawa et al.1991 , Perdigon at al. 1993 , Portier et al., 1993 , Tejada-Simon at al. 1999 , Thoreux et al. 1998 ). Our preliminary experiments require further investigation with both a nonfermented milk control group and a middle-aged group.

Our study suggests that kefir, like other fermented milk products, may stimulate mucosal immunity, although the mechanism(s) has not been resolved. The general consensus suggests that probiotic organisms must be ingested continuously to realize their health benefits (Fuller 1991 ). Furthermore, viable probiotic organisms may not be required to enhance the immune response. Improved lactose digestion and the modulation of certain immune activities have been reported with nonviable probiotic organisms (Sanders 2000 ). Unfortunately, we have no information concerning the survivability of the yeast and LAB strains present in the kefir grains employed in this study.

Differences in the genus, species or strain of probiotic bacteria can contribute to differences in traits such as stability, enzyme expression, carbohydrate fermentation patterns, acid production, colonizing ability and, perhaps most important, clinical efficacy. The composition of kefir varies dramatically depending on a variety of factors, including the source of the milk, its fat content and the composition of the grains or starters. Kefir grains include LAB (lactobacilli, lactocci, leuconostocs), yeasts, acetic acid bacteria and possibly other microorganisms (Marshall and Cole 1985 ). The predominant lactobacilli in kefir grains are L. Paracasei subsp. paracasei, L. acidophilus, L. delbrueckii subsp. Bulgaricus, L. plantarum and L. kefiranofaciens (Hallé et al. 1994 ). These strains account for 90% of the population in the grains, but only 20% of the lactobacilli in the final fermented beverage. The remaining 80% of these LAB consists of L. kefir. The predominant yeasts in both the beverage and the kefir grains are Saccharomyces cerevisiae, S. unisporus, Candida kefir and Kluyveromyces marxianus marxianus (Hallé et al. 1994 ). Although the complexity of the bacterial and yeast populations in the kefir grains, as well as in the kefir milk, precludes the identification of a specific probiotic agent responsible for immunomodulation, one possible source of adjuvant activity may be bacterial wall components.

Preservation of the normal intestinal flora, resistance to colonization and the production of antibacterial substances are critical events in the generation of a successful probiotic effect in the intestine. Despite a lack of knowledge concerning the mechanism(s) whereby probiotic organisms elicit their beneficial effects, the consumption of yogurt and fermented milks has increased in recent years and reflects perceived health benefits (Sanders 2000 ). The results of our study show that orally administered kefir enhances the specific intestinal mucosal immune response against cholera holotoxin in young adult, but not senescent rats. Subsequent investigations should address the mechanisms responsible for the suspected health benefits of probiotic foodstuffs, as well as the credibility and suitability of such dietary supplements for enhancing the intestinal immune response to vaccination in immunocompromised individuals.

	ACKNOWLEDGMENTS

 
The authors thank Rose Wang, Cell Biology and Aging Section Laboratory (San Francisco, CA) for technical assistance and Jérôme Mengaud for kefir preparation (Centre International de Recherche Daniel Carasso, Le Plessis-Robinson, France).

	FOOTNOTES

 
¹ Supported by grants from the Department of Veterans Affairs Medical Center, San Francisco, CA, and the Danone Group, Le Plessis-Robinson, France.

³ Abbreviations used: CT, cholera toxin; ELISPOT, enzyme-linked immunospot; Ig, immunoglobulin; LAB, lactic acid bacteria; LP, lamina propria; MLN, mesenteric lymph nodes; PP, Peyer’s patches; SP, spleen.

Manuscript received October 19, 2000. Initial review completed October 27, 2000. Revision accepted December 4, 2000.

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