Immunol Allergy Clin N Am

24 (2004) 739 – 752

Probiotics and down-regulation of the

allergic response
Marko A. Kalliom7ki, MD, PhDa,*, Erika Isolauri, MD, PhDb
a
Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital,
Charlestown, MA 02129, USA
b
Department of Pediatrics, University of Turku and Turku University Hospital, P.O. Box 52,
FIN-20521, Turku, Finland

Allergy, manifested in atopic eczema, allergic rhinitis, and asthma, is the most
common chronic disease in the westernized world. The prevalence of allergic
diseases seems to be increasing in developing and developed countries [1,2]. The
steep increase in prevalence that occurred in the 20th century has been attributed
to changes in environmental factors. The hygiene hypothesis of allergy suggests
that a lack of exposure to microbes early in childhood is a major factor in this
trend [3]. Studies have demonstrated that certain strains of gut microbiota possess
immunomodulatory properties that might be advantageous when combating
allergic diseases [4]. This article focuses on probiotics and their role in regulation
of allergic response.

Probiotics: rationale for use and general criteria

Epidemiologic, experimental, and in vitro evidence supports the concept that

the increased prevalence of atopic diseases is related to reduced exposure to

The Academy of Finland and the Finnish Pediatric Research Foundation are acknowledged for
the financial support.
* Corresponding author. Department of Pediatrics, University of Turku and Turku University
Hospital, P.O. Box 52, FIN-20521, Turku, Finland.
E-mail address: marko.kalliomaki@utu.fi (M.A. Kalliom7ki).

0889-8561/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.iac.2004.06.006
740 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

microbes at an early age as a result of improved public health and living

conditions [3]. The modern diet also has been estimated to contain several
thousand times less bacteria than earlier diets. This drastic reduction in dietary
bacteria, mainly the genera Lactobacillus and Bifidobacterium, has resulted from
changes in methods of food production and preservation [5]. Because of
extensive hygiene measures practiced in medicine and daily life, children may
harbor less protective indigenous gut microbiota than in earlier times. Studies
have demonstrated that the prevalence of atopic diseases in children from families
following an anthroposophic way of life is significantly lower than in children of
families not following this lifestyle [6]. This reduced risk has been connected to
characteristics of the anthroposophic lifestyle, which include restrictive use of
antibiotics, antipyretics, and vaccinations but ample use of an organic diet (often
consisting of vegetables that spontaneously were fermented by lactobacilli) and
birth at home [6,7]. These specific lifestyle features have been shown to influence
the composition of the gut microbiota, suggesting the importance of gut
microbiota in the development of allergy [7].
Establishment of gut microbiota is a step-wise, well-controlled process that
commences after birth [8]. All infants initially are colonized by Escherichia coli
and streptococci, followed by the anaerobic genera Bacteroides, Bifidobacterium,
and Clostridium at the end of the first week of life [8]. Formula-fed infants harbor
the mixture of these strains in their gut, whereas bifidobacteria dominate in
breast-fed infants [9,10]. Breast milk, which is likely a source of lactic acid
bacteria [11], promotes that dominance. After weaning, an adult-type pattern of
intestinal micriobiota gradually becomes established [8].
Sepp et al [12] found that fecal microbiota differed significantly among
Estonian and Swedish healthy infants. The major differences were that Estonian
infants had high counts of lactobacilli and eubacteria, and Swedish infants had
increased numbers of clostridia [12]. The same research group demonstrated in a
subsequent study that 2-year old allergic children were colonized with lactobacilli
less often than were nonallergic children, but nonallergic children had higher
counts of coliforms and Staphylococcus aureus [13]. Preschool and school-aged
Japanese children with atopic eczema were shown to have lower counts of fecal
Bifidobacterium than did healthy subjects, and they also were colonized more
often with Staphylococcus [14]. A dissection of Bifidobacterium microbiota in a
Finnish study uncovered that allergic infants had an adult-like Bifidobacterium
microbiota, whereas healthy infants were colonized with a typical infant
Bifidobacterium microbiota [15]. In vitro, the adult-like bifidobacteria induced
more proinflammatory cytokines and fewer anti-inflammatory cytokines than did
the infant-like bifidobacteria, suggesting that certain Bifidobacterium species
may have antiallergic properties [16].
Two prospective studies demonstrated alterations in gut microbiota in children
who later developed IgE-mediated allergic hypersensitivity and atopic eczema.
Using fluorescence in situ hybridization, the authors’ group showed that children
who developed reactivity to skin prick tests with environmental antigens by 1 year
of age had higher counts of clostridia and lower counts of bifidobacteria in
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 741

their neonatal feces than did children who did not develop such reactivity [17].
These differences had disappeared by the age of 3 months. Using culture-
dependent methods, Bjfrksten et al [18] found that children who developed skin
prick test reactivity or atopic eczema by 2 years of age were colonized less often
with bifidobacteria during the first year of life than were children who did not
develop these disorders. These groups also exhibited differences in the colo-
nization of enterococci, clostridia, S aureus, and Bacteroides [18]. These studies
suggest that certain species of gut microbiota may contribute to the development
of a nonallergic immune system.
Probiotics are defined as live microbial food ingredients that beneficially
affect host health. They most often belong to the genera Bifidobacterium or
Lactobacillus [8,19]. Certain species of these two genera have been a part of the
human diet for millennia in the form of fermented food products [5,19]. Scientific
interest in these bacteria was raised by the Russian Nobel laureate Elie
Metchnikoff a century ago [20]. The term probiotic was introduced by Lilly and
Stillwell 3 decades ago to describe any organism or substance which contributes
to the intestinal microbial balance in animals [21]. Because of increased scientific
data concerning probiotics, the definition of the term has evolved through the
years [4].
Several criteria must be fulfilled before a bacterial strain can be regarded as a
probiotic. These factors include human origin; survival in the gut; ability to
adhere temporarily to the intestinal epithelium and to induce an immune
response; safe use in humans; scientifically documented beneficial effects; and
invariable properties during all stages of manufacturing, processing, and
preservation [4,8]. The latter criterion also includes administration (ie, native
beneficial strains of the gut microbiota are not probiotics unless they have been
isolated, purified, characterized, evaluated, and used as probiotics) [19]. Pro-
biotics must demonstrate measurable clinical health benefits [19]. All candidate
probiotic strains should be evaluated closely, and beneficial effects of a cer-
tain strain should not be related to any other strain without indisputable proof
[4,8,19]. Attachment to intestinal mucus, a critical component in a cascade lead-
ing to an immune response, reliably can be evaluated using endoscopic biopsies,
which have been shown to be superior to fecal samples in this regard [22].
Because of vigorous research in the field, these criteria should be under
continuous critical evaluation. Even the need of oral administration has been
challenged in a study addressing the role of probiotics in prevention and treat-
ment of experimental colitis [23].
Safety is a critical issue in the use of any potential therapeutic intervention.
According to one review, oral consumption of lactobacilli and bifidobacteria
by healthy people did not increase risk for bacterial diseases [24]. Even in
immunocompromised patients, the risk for severe bacterial infection is low [25].
Salminen et al [25] evaluated the clinical data from 89 patients with
Lactobacillus-induced bacteremia. In 11 patients, the isolated strain was identical
to the probiotic Lactobacillus rhamnosus GG. Cases of Lactobacillus-induced
bacteremia usually were associated with severe underlying comorbidities [25].
742 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

Regulation of allergic inflammation: more than a mere Th1–Th2 paradigm

A T helper cell type 2 (Th2)-skewed immune response is a hallmark of allergic

immune responses and atopic disease in the gut and other target organs [26,27].
Th2 cells produce several cytokines and chemokines that amplify allergic inflam-
mation, including interleukin 4 (IL-4) and IL-5, which increase IgE synthesis and
eosinophil growth and differentiation, respectively. Mast cell differentiation,
enhanced by IL-9, and airway hyperreactivity, induced by IL-13, are other
cardinal features of the allergic response that is regulated by Th2 cells [26].
Initially, however, all T-cell responses to environmental antigens are Th2 oriented
[28]. That kind of natural immune response is a necessity for the maintenance of
successful pregnancy [29]. On postnatal microbial stimulation, a new Th1–Th2
balance is established [30]. With an inappropriate stimulus, an allergic type of
immune responsiveness may ensue [30].
Cytokines produced by Th1 and Th2 cells are capable of cross-regulating each
other’s development. Once either kind of response has been established firmly,
the cross-regulation is more difficult [26]. Experimental studies suggested that
Th1 cells may exacerbate rather than alleviate a Th2-skewed allergic inflamma-
tory response [31]. Other T cells, in addition to those involved in the Th1–Th2
paradigm, are important in the regulation of the allergic response. These
regulatory T cells include Th3 cells, T regulatory 1 cells, CD4+ CD25+ T cells,
and natural killer T (NKT) cells [32].
Transforming growth factor b (TGF-b), the principal cytokine of Th3 cells,
reversed allergen-induced airway hyperreactivity and inflammation in mice [33].
High concentrations of TGF-b in breast milk were associated with prevention of
early atopic eczema in breast-fed infants [34]. IL-10, the most abundantly
expressed cytokine of T regulatory 1 cells, prevented asthmatic inflammation and
food allergy in two different mouse models [35,36]. IL-10 was induced in the
latter study by enteric helminth infection. In parallel, parasite-induced IL-10
production in humans has been suggested to mediate an inhibitory effect of
helminth infections on allergen skin test reactivity [37,38].
CD4+ CD25+ T cells are naturally occurring and mainly thymus-derived
regulatory cells that are involved in the maintenance of self-tolerance. Depletion
of these cells results in autoimmune diseases in animal models [32]. This finding
also has been seen in patients with X-linked autoimmunity allergic dysregula-
tion syndrome and immune-dysregulation, polyendocrinopathy, enteropathy
X-linked syndrome [39,40]. These patients lack normal CD4+ CD25+ regulatory
T cells because of genetic mutations in the transcription factor Foxp3, a criti-
cal regulator of CD4+ CD25+ T-cell development and function [39,40].
These patients also develop food allergy, severe atopic eczema, and increased
amounts of IgE and eosinophils in their serum, suggesting a regulatory control
of CD4+ CD25+ T cells in the development of allergic diseases [39,40]. One
clinical study found that the suppressive potential of CD4+ CD25+ T cells in
adults with respiratory allergies was reduced significantly, as compared with
the potential in healthy adults [41]. Allergic symptoms may result from an
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 743

inappropriate balance between regulatory CD4+ CD25+ T cells and effector

Th2 cells [41].
NKT cells are a T-cell population that expresses cell surface markers char-
acteristic of natural killer cells and T cells. Once activated, these cells produce
large amounts of Th1 and Th2 cytokines that may allow the cells to exert strong
regulatory activity in autoimmune and allergic diseases [32]. Two studies using
three different NKT cell-deficient mouse strains with Th2-skewed immune
responses have demonstrated that allergen-induced airway inflammation and
hyperreactivity are dependent on IL-4 and IL-13 produced by iVa14 NKT cells
[42,43]. The antigen-specific IgE response was impaired in these mice [43].
These studies suggest that Th2 responses alone are not sufficient and that NKT
cell-produced cytokines are needed for the development of IgE-mediated aller-
gic asthma.
The previously mentioned findings clearly suggest that an immunologic treat-
ment of choice for allergy should combine Th1-like and suppressive properties.

Effects of probiotics in clinical studies of allergy

Randomized, placebo-controlled trials of probiotics mostly have focused on

patients with atopic eczema. Majamaa and Isolauri [44] evaluated infants with
atopic eczema and cow’s milk allergy. The infants were treated with an ex-
tensively hydrolyzed whey formula with or without the addition of L rhamnosus
GG. The clinical score of atopic eczema improved significantly during the
1-month study period in infants receiving the formula plus the probiotic [44].
Concentrations of fecal a1-antitrypsin and tumor necrosis factor a, which are
markers of intestinal inflammation, decreased significantly in this group but
not in the group receiving the formula only [44]. A comparable effect on atopic
eczema was found in a clinical trial in which extensively hydrolyzed whey
formula was fortified with L rhamnosus GG or Bifidobacterium lactis Bb-12
[45]. These formulas were administered to infants who had manifested atopic
eczema during exclusive breast-feeding. In parallel, markers of allergic in-
flammation and concentrations of soluble CD4 in serum and eosinophilic protein
X in urine were reduced significantly in these patients, as compared with levels in
patients receiving the formula only [45]. In a crossover study, Rosenfeldt et al
[46] gave a combination of L rhamnosus 19070-2 and Lactobacillus reuteri DSM
122,460 to 1- to 13-year-old children with atopic eczema for 6 weeks. They
found that the combination was beneficial, especially in patients with a positive
skin prick test response and increased IgE concentrations [46], suggesting that
probiotics may be able to control inflammatory responses beyond those in the
gut. The concentration of serum eosinophil cationic protein decreased during
active treatment, although the production of the cytokines IL-2, IL-4, and IL-10
and interferon g (IFN-g) by peripheral blood mononuclear cells remained
unaltered [46]. In general, significant adverse effects have not been reported in
studies using viable probiotic preparations in allergic patients. This finding may
744 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

not occur in studies using nonviable probiotics, because a pilot study addressing
the effect of heat-inactivated L rhamnosus GG on atopic eczema was terminated
early because of adverse gastrointestinal symptoms [47].
Two randomized, placebo-controlled clinical studies have evaluated the effects
of lactobacilli on respiratory allergic diseases. Wheeler et al [48] conducted a
crossover trial with yogurt containing Lactobacillus acidophilus in adult patients
with moderate asthma. They found that consumption of this yogurt containing
tended to increase IFN- g production in stimulated lymphocytes and decrease
levels of eosinophilia, although no changes in clinical asthma parameters were
detected [48]. No beneficial treatment effect of L rhamnosus GG was found in
teenagers and young adults who were allergic to birch pollen and had intermittent
respiratory symptoms [49].
The authors previously have addressed whether probiotics might prevent early
atopic disease. In one study, L rhamnosus GG was administered perinatally to
mothers and infants up to 6 months of age [50]. Children had a family history of
atopic disease. The administration of the probiotic halved the incidence of atopic
eczema during the first 2 years of life. There were no differences in IgE-mediated
allergic hypersensitivity as measured by skin prick test reactivity and serum IgE
concentrations [50]. The extension of the preventive effect beyond infancy was
demonstrated in the 4-year follow-up of the study cohort [51]. Consumption of
L rhamnosus GG by pregnant and lactating mothers increased the amount of
TGF-b in breast milk. The risk for atopic eczema during infancy was reduced
among infants whose mothers consumed the probiotic strain [52].

Effects of probiotics in experimental studies of allergy

The impact of probiotics on allergic diseases has been studied in a few animal
models. Lactobacillus plantarum L-137 was shown to inhibit antigen-specific
IgE production in DBA/2 mice that were fed a casein diet. The suppressive effect
of the strain was related to enhanced production of IL-12, an inducer of the Th1
response, by peritoneal macrophages [53]. The Lactobacillus casei strain Shirota
was found to suppress IgE and IgG1 responses and systemic anaphylaxis in a
food allergy model with ovalbumin-specific T-cell–receptor transgenic mice. The
effect was mediated by induction of IL-12 production [54]. In both of these
studies, probiotics were administered by intraperitoneal injection.
In two murine models, Sudo et al [55] studied the effects of antibiotic-induced
alterations of gut microbiota and probiotic supplementation on the development
of Th2-skewed immune response. BALB/c mice who were treated with
kanamycin during the neonatal period developed a Th2-skewed immune
response. The change was reversed by oral introduction of Enterococcus faecalis
or L acidophilus (the latter to a lesser extent) [55]. Inoculation with Bacteroideus
vulcatus increased the Th2-skewed immune response. C57BL/6 mice that
genetically are biased toward Th1-immunity were resistant to kanamycin
treatment [55].
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 745

Prioult et al [56] compared the abilities of three probiotics (ie, Lactobacillus

paracasei, Lactobacillus johnsonii, Bifidobacterium lactis Bb12) to induce and
maintain oral tolerance to b-lactoglobulin in germfree mice. Humoral and cellular
immune responses were suppressed most in L paracasei-monoassociated mice,
although not as much as in conventionally colonized mice [56]. These findings
suggest that probiotic intervention may help oral-tolerance induction and prevent
Th2-shifted immune responses, and that genetic background contributes to
immune deviation.
Two experimental studies have evaluated effects of intranasal co-application
of probiotics and allergen on immune responses. Kruisselbrink et al [57] con-
structed a recombinant L plantarum strain that expressed an immunodomi-
nant T-cell epitope of the Der p 1 allergen of the house dust mite. Intranasal
application of this construct to Der p 1-immunized mice induced suppression of
the Th1 and Th2 immune responses that were elicited by the antigen [57]. When
intranasally co-applied with recombinant Bet v 1, the major birch pollen aller-
gen (Lactococcus lactis and L plantarum) increased levels of IgG2a antibodies,
in vitro IFN-g production, and suppression of allergen-induced basophil
degranulation [58]. These studies indicate that certain probiotic strains, when
combined with allergens, are potential candidates for mucosal vaccination
against allergy.

Antiallergic properties of probiotics: lessons from in vitro studies

Food and other orally consumed products, including probiotics, are subjected
to gastrointestinal characteristics, including peristalsis, gastric juice, bile, and
digestive enzymes. These factors assist in digestion and down-regulation of the
immune response because without degradation unresponsiveness to dietary
antigens is not achieved [59]. L rhamnosus GG has been found to enhance the
degradation of dietary antigens in rats [60]. Bovine casein degraded with
L rhamnosus GG-derived proteases was shown to suppress lymphocyte prolifera-
tion in a dose-dependent manner and reduce the anti-CD3–stimulated IL-4
production by peripheral blood mononuclear cells in children with atopic eczema
[61,62]. In one study, homogenates derived from B lactis, L acidophilus, and
Lactobacillus delbrueckii subspecies bulgarius (but not from Streptococcus
thermophilus) suppressed proliferation of human mononuclear cells in vitro,
although not as much as L rhamnosus GG did [63]. Bovine casein degraded with
L rhamnosus GG-derived proteases suppressed T-cell activation by inhibition
of protein kinase C activation [64]. Activation of protein kinase C is an
indispensable stage in the nuclear factor kB (NF-kB) activation cascade in T cells,
ultimately leading to the expression of gene products that mediate innate and
adaptive immunity [65]. These studies suggest that certain probiotic strains may
contribute to the processing of dietary antigens in the intestinal lumen in such a
way as to reduce their immunogenicity by suppressing T-cell activation.
746 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

Probiotics have been shown to amplify the gut mucosal barrier. L rhamnosus
GG and L plantarum 299v, but not L acidophilus DDS-1, adhered effectively to
HT 29 intestinal epithelial cells and increased expression of MUC2 and MUC3
intestinal mucins [66,67]. This up-regulation of mucins inhibited the adherence of
E coli to the cell line [66,67]. This finding may be important in allergy, because
total serum IgE concentration correlated directly with fecal E coli counts in
infants with early onset food-allergy and atopic eczema [68]. Supplementation
with B lactis Bb-12 resulted in decreased numbers of E coli, as measured by
fluorescence in situ hybridization [68]. L rhamnosus GG reversed the increased
intestinal permeability that was induced by cow’s milk in suckling rats [69]. The
permeability was measured by absorption of horseradish peroxidase across
segments containing Peyer’s patches and patch-free jejunal segments [69]. The
same method was used to show that antigen transfer across the gut mucosal
barrier is increased in children with atopic eczema [70]. In theory, by reducing
antigen transfer to the lamina propria, probiotics might reduce the quantity and
quality of the antigenic load on the gut-associated lymphatic tissue in children
with atopic eczema.
Beyond physical support, a single layer of epithelial cells between the
intestinal lumen and lamina propria constitutes a major compartment of the gut
immune system [71]. Intestinal epithelial cells possess delicate mechanisms that
allow symbiotic coexistence with abundant resident luminal bacteria while
maintaining alert responsiveness to enteropathogenic intruders [71–73]. Lacto-
bacilli are likely to maintain homeostasis in intestinal epithelial cells. Treatment
with L johnsonii La1, L acidophilus La10, or lipoteichoic acids from these
stains did not result in a proinflammatory response in a co-culture with human
intestinal HT29 cells [74]. The lipoteichoic acids, however, antagonized the
proinflammatory response of HT29 cells induced by lipopolysaccharide and
gram-negative bacteria [74]. L rhamnosus GG prevented cytokine-induced
apoptosis in mouse and human colon cells [75]. This effect was mediated by
activation of anti-apoptotic Akt and protein kinase B, a part of the phospho-
inositide 3-kinase pathway, and inactivation of the pro-apoptotic p38 mitogen-
activated protein kinase (p38 MAPK) signaling pathway [75]. p38 MAPK
and NF-kB pathways are downstream of a common myloid differention
factor 88-dependent signaling pathway [76]. p38 MAPK, a novel target of anti-
inflammatory therapies, has been linked to a wide range of inflammatory re-
sponses in the gut [77]. L acidophilus and L casei also stimulated Akt activation
but did not inactivate the p38 MAPK pathway [75]. These findings indicate
that lactobacilli may have strain-specific anti-inflammatory effects in the intesti-
nal epithelium.
Dendritic cells have a pivotal role in orchestration of the immune system [78].
In the gastrointestinal tract, they reside in the Peyer’s patches, lamina propria, and
draining mesenteric lymph nodes, representing the principal stimulators of naRve
T cells [79]. Intestinal dendritic cells can sample microbes and other intestinal
antigens by way of M cells in Peyer’s patches, directly from the intestinal lumen
by reaching between adjacent epithelial cells and indirectly by sampling
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 747

apoptotic epithelial cells or taking up antigen-bearing exosomes. Direct contact

with antigens is also possible when epithelial integrity is disrupted [79]. The fate
of a naRve T cell is determined by three dendritic cell-derived signals: antigen
(signal 1), co-stimulation (signal 2), and cytokines (signal 3) [78]. The expression
of different toll-like receptors (TLRs) in antigen-presenting cells allows the cells
to discriminate between specific patterns of microbial products found in patho-
genic and nonpathogenic strains [80]. E coli LPS is recognized by TLR4,
whereas TLR2 recognizes peptidoglycans of S aureus. In parallel, lipoteichoic
acids from lactobacilli are recognized by TLR2 [81]. On recognition of their
ligands, TLRs initiate signaling processes that lead to maturation of dendritic
cells and secretion of cytokines and chemokines by the cells. These cell products
influence the polarization of T helper cells and greatly regulate adaptive immune
responses [78]. All of the six lactobacilli studied were capable of maturation of
the bone marrow-derived murine dendritic cells in vitro [82]. L casei CHCC3139
was the most potent IL-12 (Th1) inducer, and Lactobacillus reuteri DSM12246
the weakest inducer [82]. In co-culture, however, the latter strain prevented
dendritic cell maturation and the production of IL-12, IL-6 and tumor necrosis
factor a that had been induced by the former strain [82]. Braat et al [83]
compared the abilities of L rhamnosus GG and the intestinal commensal
Klebsiella pneumoniae to induce maturation and polarization of human dendritic
cells. Both bacterial strains induced dendritic cell maturation, although
K pneumoniae-matured dendritic cells produced much more Th1 cytokines than
did the cells matured by L rhamnosus GG [83]. In parallel, naive T cells primed
with K pneumoniae-matured dendritic cells adopted a Th1 phenotype and
produced also IL-10, whereas T cells generated from L rhamnosus-matured
dendritic cells produce equal amounts of IL-4 and IFN-g (Th0 phenotype). These
cells also produced some IL-10 [83], suggesting that they might have suppressive
properties. Ingestion of L rhamnosus GG has been shown to result in elevation of
serum IL-10 concentrations in children with atopic eczema, as compared with
children receiving placebo [84]. The increase of mitogen-induced IL-10
production by peripheral blood mononuclear cells preceded the increase in
serum IL-10 levels [84], a finding which indirectly suggests that the previously
mentioned dendritic cell-driven mechanism might also function in vivo. These
findings indicate that lactobacilli may be able to induce the maturation of
dendritic cells that prime T cells to produce mainly Th1 and regulatory cytokines.
Probiotics exert mostly proinflammatory immune responses when co-cultured
with peripheral blood mononuclear cells or macrophages. Miettinen et al [85] and
Hessle et al [86] studied the effects of six Lactobacillus strains in co-culture with
human peripheral blood mononuclear cells. Five strains elicited strong IL-12,
minor IL-10, and no IL-4 production, suggesting Th1-oriented immune responses
[85,86]. L paracasei NCC2461, however, induced production of suppressive
IL-10 and TGF-b in a mixed culture of murine splenocytes and CD4+ T cells [87].
L rhamnosus GG induced Th1 chemokine production in human macrophages
[88], and the same strain has been shown to activate the transcription factor NF-
kB directly and to activate STAT1 and STAT3 indirectly by way of cytokines
748 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

Table 1
Potential antiallergic properties of probiotics
Site of action Effects
Gastrointestinal mucus Processing of enteral antigens
and epithelium Inhibition of attachment of pathogenic bacteria
Amplification of permeability barrier
Suppression of proinflammatory responses elicited
by pathogenic bacteria
Prevention of apoptosis
Lamina propria Maturation of dendritic cells with Th1-type
and suppressive properties
Production of Th1 and suppressive cytokines
by myeloid-derived immune cells

[89]. These chemokines were shown to stimulate Th1 cell chemotaxis [88].
Studies with murine macrophages suggest that lactobacilli induce their effect on
these cells, at least partially, by way of TLR2 that is stimulated by lipoteichoic
acids [81].

Summary

The first clinical trials with probiotics, especially in the treatment of atopic
eczema, have yielded encouraging results. Experimental studies have found that
probiotics exert strain-specific effects in the intestinal lumen and on epithelial
cells and immune cells with anti-allergic potential. These effects include
enhancement in antigen degradation and gut barrier function and induction of
regulatory and proinflammatory immune responses, the latter of which occurs
more likely beyond the intestinal epithelium (Table 1). Future studies should
address more accurately how these and other possible mechanisms operate in the
complex gastrointestinal macroenvironment in vivo and how these mechanisms
are related to the clinical effects in a dose-dependent manner.

References

[1] Holgate ST. The epidemic of allergy and asthma. Nature 1999;402(6760 Suppl):B2 – 4.
[2] Wickman M, Lilja G. Today, one child in four has an ongoing allergic disease in Europe: what
will the situation be in tomorrow? Allergy 2003;58(7):570 – 1.
[3] Prescott SL. Allergy: the price we pay for cleaner living? Ann Allergy Asthma Immunol 2003;
90(6 Suppl 3):64 – 70.
[4] Isolauri E, Rautava S, Kalliom7ki M, Kirjavainen P, Salminen S. Role of probiotics in
food hypersensitivity. Curr Opin Allergy Clin Immunol 2002;2(3):263 – 71.
[5] Bengmark S. Ecological control of the gastrointestinal tract: the role of probiotic flora. Gut
1998;42(1):2 – 7.
[6] Alm JS, Swartz J, Lilja G, Scheynius A, Pershagen G. Atopy in children of families with
an anthroposophic lifestyle. Lancet 1999;353(9163):1485 – 8.
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 749

[7] Alm JS, Swartz J, Bjfrksten B, Engstrand L, Engstrfm J, Kqhn I, et al. An anthroposophic
lifestyle and intestinal microflora in infancy. Pediatr Allergy Immunol 2002;13(6):402 – 11.
[8] Salminen S, Bouley C, Boutron-Ruault MC, Cummings JH, Frank A, Gibson GR, et al.
Functional food science and gastrointestinal physiology and function. Br J Nutr 1998;
80(Suppl 1):147 – 71.
[9] Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, et al.
Analysis of intestinal flora development in breast-fed and formula-fed infants by using
molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000;30(1):61 – 7.
[10] Favier CF, Vaughan EE, De Vos WM, Akkermans ADL. Molecular monitoring of succession
of bacterial communities in human neonates. Appl Environm Microbiol 2002;68(1):219 – 26.
[11] Martin R, Langa S, Reviriego C, Jimenez E, Marin ML, Xaus J, et al. Human milk is a source
of lactic acid bacteria. J Pediatr 2003;143(6):754 – 8.
[12] Sepp E, Julge K, Vasar M, Naaber P, Bjfrksten B, Mikelsaar M. Intestinal microflora
of Estonian and Swedish infants. Acta Paediatr 1997;86(9):956 – 61.
[13] Bjfrksten B, Naaber P, Sepp E, Mikelsaar M. The intestinal microflora in allergic Estonian
and Swedish 2-year-old children. Clin Exp Allergy 1999;29(3):342 – 6.
[14] Watanabe S, Narisawa Y, Arase S, Okamatsu H, Ikenaga T, Tajiri Y, et al. Differences in
fecal microflora between patients with atopic dermatitis and healthy control subjects. J Allergy
Clin Immunol 2003;111(3):587 – 91.
[15] Ouwehand AC, Isolauri E, He F, Hashimoto H, Benno Y, Salminen S. Differencies in
Bifidobacterium flora composition in allergic and healthy infants. J Allergy Clin Immunol 2001;
108(1):144 – 5.
[16] He F, Morita H, Ouwehand AC, Hosoda M, Hiramatsu M, Kurisaki J, et al. Stimulation of
the secretory pro-inflammatory cytokines by Bifidobacterium strains. Microbiol Immunol
2002;46(11):781 – 5.
[17] Kalliom7ki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E. Distinct patterns of
neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin
Immunol 2001;107(1):129 – 34.
[18] Bjfrksten B, Sepp E, Julge K, Voor T, Mikelsaar M. Allergy development and the intestinal
microflora during the first year of life. J Allergy Clin Immunol 2001;108(4):516 – 20.
[19] Sanders ME. Probiotics: considerations for human health. Nutr Rev 2003;61(3):91 – 9.
[20] Metchnikoff E. The prolongation of life: optimistic studies. London7 William Heinemann; 1907.
[21] Lilly DM, Stillwell RH. Probiotics: growth-promoting factors produced by microorganisms.
Science 1965;147:747 – 8.
[22] Alander M, Satokari R, Korpela R, Saxelin M, Vilpponen-Salmela T, Mattila-Sandholm T, et al.
Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus
rhamnosus GG, after oral consumption. Appl Environ Microbiol 1999;65(1):351 – 4.
[23] Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky B, et al. Toll-like
receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental
colitis. Gastroenterology 2004;126(2):520 – 8.
[24] Salminen S, von Wright A, Morelli L, Marteau P, Brassart D, de Vos WM, et al. Demonstration
of safety of probiotics—a review. Int J Food Microbiol 1998;44(1–2):93 – 106.
[25] Salminen MK, Rautelin H, Tynkkynen S, Poussa T, Saxelin M, Valtonen V, et al. Lactobacillus
bacteremia, clinical significance, and patient outcome, with special focus on probiotic
L. Rhamnosus GG. Clin Infect Dis 2004;38(1):62 – 9.
[26] Romagnani S. The role of lymphocytes in allergic disease. J Allergy Clin Immunol
2000;105(3):399 – 408.
[27] Brandt EB, Strait RT, Hershko D, Wang Q, Muntel EE, Scribner TA, et al. Mast cells are
required for experimental oral allergen-induced diarrhea. J Clin Invest 2003;112(11):1666 – 77.
[28] Prescott SL, Macaubas C, Holt BJ, Smallacombe TB, Loh R, Sly PD, et al. Transplacental
priming of the human immune system to environmental allergens: universal skewing of initial
T cell responses toward the Th2 cytokine profile. J Immunol 1998;160(10):4730 – 7.
[29] Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of
750 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

both leukaemia inhibitory factor and type 2 T helper cytokines by decidual T cells in
unexplained recurrent abortions. Nat Med 1998;4(9):1020 – 4.
[30] Prescott SL, Macaubes C, Smallacombe T, Holt BJ, Sly PD, Holt PG. Development of allergen-
specific T cell memory in atopic and normal children. Lancet 1999;353(9148):196 – 200.
[31] Hansen G, Berry G, DeKruyff RH, Umetsu DT. Allergen-specific Th1 cells fail to counter-
balance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J Clin
Invest 1999;103(2):175 – 83.
[32] Umetsu DT, Akbari O, Dekruyff RH. Regulatory T cells control the development of allergic
disease. J Allergy Clin Immunol 2003;112(3):480 – 7.
[33] Hansen G, McIntire JJ, Yeung VP, Berry G, Thorbecke GJ, Chen L, et al. CD4(+) T helper
cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity
and inflammation. J Clin Invest 2000;105(1):61 – 70.
[34] Kalliom7ki M, Quwehand A, Arvilommi H, Kero P, Isolauri E. Transforming growth factor-beta
in breast-milk: a potential regulator of atopic disease at an early age. J Allergy Clin Immunol
1999;104(6):1251 – 7.
[35] Oh JW, Seroogy CM, Meyer EH, Akbari O, Berry G, Fathman CG, et al. CD4 T helper cells
engineered to produce IL-10 prevent allergen-induced airway hyperreactivity and inflammation.
J Allergy Clin Immunol 2002;110(3):460 – 8.
[36] Bashir ME, Andersen P, Fuss IJ, Shi HN, Nagler-Anderson C. An enteric helminth infection
protects against an allergic response to dietary antigen. J Immunol 2002;169(6):3284 – 92.
[37] van den Biggelaar AHJ, van Ree R, Rodrigues LC, Lell B, Deelder AM, Kremsner PG, et al.
Decreased atopy in children infected with Schistosoma haematobium: a role for parasite
induced interleukin-10. Lancet 2000;356(9243):1723 – 7.
[38] Cooper PJ, Chico ME, Rodrigues LC, Ordonez M, Strachan D, Griffin GE. Reduced risk of
atopy among school-age children infected with geohelminth parasites in a rural area of the
tropics. J Allergy Clin Immunol 2003;111(5):995 – 1000.
[39] Chatila TA, Blaeser F, Ho N, Lederman HN, Voulgaropoulos C, Helms C, et al. JM2, encoding
a fork head related protein, is mutated in X-linked autoimmunity-allergic disregulation
syndrome. J Clin Invest 2000;106(12):R75 – 81.
[40] Owen CJ, Jennings CE, Imrie H, Lachaux A, Bridges NA, Cheetham TD, et al. Mutational
analysis of the FOXP3 gene and evidence for genetic heterogeneity in the immunodysregulation,
polyendocrinopathy, enteropathy syndrome. J Clin Endocrinol Metab 2003;88(12):6034 – 9.
[41] Ling EM, Smith T, Dao Nguyen X, Pridgeon C, Dallman M, Arbery J, et al. Relation of
CD4 + CD25 + regulatory T-cell suppression of allergen-driven T-cell activation to atopic
status and expression of allergic disease. Lancet 2004;363(9409):608 – 15.
[42] Akbari O, Stock P, Meyer E, Kronenberg M, Sidobre S, Nakayama T, et al. Essential role of
NKT cells producing IL-4 and IL-13 in the development of allergen induced airway
hyperreactivity. Natl Med 2003;9(5):582 – 8.
[43] Lisbonne M, Diem S, de Castro Keller A, Lefort J, Araujo LM, Hachem P, et al. Cutting edge:
V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity
in an experimental asthma model. J Immunol 2003;171(4):1637 – 41.
[44] Majamaa H, Isolauri E. Probiotics: a novel approach in patients with food allergy. J Allergy
Clin Immunol 1997;99(2):179 – 85.
[45] Isolauri E, Arvola T, Sqtas Y, Moilanen E, Salminen S. Probiotics in the management of
atopic eczema. Clin Exp Allergy 2000;30(11):1604 – 10.
[46] Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppesen DL, Valerius NH, et al.
Effect of probiotic Lactobacillus strains in children with atopic dermatitis. J Allergy Clin
Immunol 2003;111:389 – 95.
[47] Kirjavainen PV, Salminen SJ, Isolauri E. Probiotic bacteria in the management of atopic disease:
underscoring the importance of viability. J Pediatr Gastroenterol Nutr 2003;36(2):223 – 7.
[48] Wheeler JG, Shema SJ, Bogle ML, Shirrell MA, Burks AW, Pittler A, et al. Immune and clinical
impact of Lactobacillus acidophilus on asthma. Ann Allergy Asthma Immunol 1997;79(3):
229 – 33.
[49] Helin T, Haahtela S, Haahtela T. No effect of oral treatment with an intestinal bacteria strain,
 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752 751

Lactobacillus rhamnosus (ATCC 53103), on birch-pollen allergy: a placebo-controlled double-

blind study. Allergy 2002;57(3):243 – 6.
[50] Kalliom7ki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in the
primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001;
357(9262):1076 – 9.
[51] Kalliom7ki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and prevention of
atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet 2003;
361(9372):1869 – 71.
[52] Rautava S, Kalliom7ki M, Isolauri E. Probiotics during pregnancy and breast-feeding
might confer immunomodulatory protection against atopic disease in the infant. J Allergy Clin
Immunol 2002;109(1):119 – 21.
[53] Murosaki S, Yamamoto Y, Ito K, Inokuchi T, Kusaka H, Ikeda H, et al. Heat-killed
Lactobacillus plantarum L-137 suppresses naturally fed antigen-specific IgE production by
stimulation of IL-12 production in mice. J Allergy Clin Immunol 1998;102(1):57 – 64.
[54] Shida K, Takahashi R, Iwadate E, Takamizawa K, Yasui H, Sato T, et al. Lactobacillus casei
strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and
systemic anaphylaxis in a food allergy model. Clin Exp Allergy 2002;32(4):563 – 70.
[55] Sudo N, Yu X-N, Aiba Y, Oyama N, Sonoda J, Koga Y, et al. An oral introduction of intes-
tinal bacteria prevent the development of a long-term Th2-skewed immunological memory
induced by neonatal antibiotic treatment in mice. Clin Exp Allergy 2002;32(7):1112 – 6.
[56] Prioult G, Fliss I, Pecquet S. Effect of probiotic bacteria on induction and maintenance of oral
tolerance to beta-lactoglobulin in gnotobiotic mice. Clin Diag Lab Immunol 2003;10(5):787 – 92.
[57] Kruisselbrink A, Heijne Den Bak-Glashouwer MJ, Havenith CEG, Thole JER, Janssen R.
Recombinant Lactobacillus plantarum inhibits house dust mite-specific T-cell responses. Clin
Exp Immunol 2001;126(1):2 – 8.
[58] Repa A, Grangette C, Daniel C, Hochreiter R, Hoffmann-Sommergruber K, Thalhamer J, et al.
Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory
immune responses in a murine model of birch pollen allergy. Vaccine 2003;22(1):87 – 95.
[59] Barone KS, Reilly MR, Flanagan MP, Michael JG. Abrogation of oral tolerance by feeding
encapsulated antigen. Cell Immunol 2000;199(2):65 – 72.
[60] Pessi T, Sqtas Y, Marttinen A, Isolauri E. Probiotics reinforce mucosal degradation of
antigens in rats: implications for therapeutic use of probiotics. J Nutr 1998;28(12):2313 – 8.
[61] Sqtas Y, Soppi E, Korhonen H, Syv7oja EL, Saxelin M, Rokka T, et al. Suppression of
lymphocyte proliferation in vitro by bovine caseins hydrolysed with Lactobacillus casei
GG-derived enzymes. J Allergy Clin Immunol 1996;98(1):216 – 24.
[62] Sqtas Y, Hurme M, Isolauri E. Down-regulation of anti-CD3 antibody-induced IL-4 production
by bovine caseins hydrolysed with Lactobacillus GG derived enzymes. Scan J Immunol 1996;
43(6):687 – 9.
[63] Pessi T, Sqtas Y, Saxelin M, Kallioinen H, Isolauri E. Antiproliferative effects of homogenates
derived from five strains of candidate probiotic bacteria. Appl Environ Microbiol 1999;
65(11):4725 – 8.
[64] Pessi T, Isolauri E, Sqtas Y, Kankaanranta H, Moilanen E, Hurme M. Suppression of T cell
activation by Lactobacillus GG-degraded bovine casein. Int Immunopharmacol 2001;1(2):
211 – 8.
[65] Tan SL, Parker PJ. Emerging and diverse roles of protein kinase C in immune cell signalling.
Biochem J 2003;376(Pt 3):545 – 52.
[66] Mack DR, Michail S, Wel S, McDougall L, Hollingsworth MA. Probiotics inhibit entero-
pathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression. Am J
Physiol 1999;276(4 Pt 1):G941 – 50.
[67] Mack DR, Ahrne S, Hyde L, Wie S, Hollingsworth MA. Extracellular MUC3 mucin secretion
follows adherence of Lactobacillus strains to intestinal epithelial cell in vitro. Gut 2003;52(6):
827 – 33.
[68] Kirjavainen PV, Arvola T, Salminen S, Isolauri E. Aberrant composition of gut microbiota
of allergic infants: a target of bifidobacterial therapy at weaning? Gut 2002;51(1):51 – 5.
752 M.A. Kalliomäki, E. Isolauri / Immunol Allergy Clin N Am 24 (2004) 739–752

[69] Isolauri E, Majamaa H, Arvola T, Rantala I, Virtanen E, Arvilommi H. Lactobacillus casei

strain GG reverses increased intestinal permeability induced by cow milk in suckling rats.
Gastroenterology 1993;105(6):1643 – 50.
[70] Majamaa H, Isolauri E. Evalution of the hut mucosal barrier: evidence for increased antigen
transfer in children with atopic eczema. J Allergy Clin Immunol 1996;97(4):985 – 90.
[71] Haller D, Jobin C. Interaction between resident luminal bacteria and the host: can a healthy
relationship turn sour? J Pediatr Gastroenterol Nutr 2004;38(2):123 – 36.
[72] Melmed G, Thomas LS, Lee N, Tesfay SY, Lukasek K, Michelsen KS, et al. Human intestinal
epithelial cells are broadly unresponsive Toll-like receptor 2-dependent bacterial ligands: impli-
cations for host-microbial interactions in the gut. J Immunol 2003;170(3):1406 – 15.
[73] Hornef MW, Normark BH, Vandewalle A, Normark S. Intracellular recognition of lipo-
polysaccharide by toll-like receptor 4 in intestinal epithelial cells. J Exp Med 2003;198(8):
1225 – 35.
[74] Vidal K, Donnet-Hughes A, Granato D. Lipoteichoic acids from Lactobacillus johnsonii Strain
La1 and Lactobacillus acidophilus La10 antagonize the responsiveness of human intestinal
epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria. Infect Immunol 2002;
70(4):2057 – 64.
[75] Yan F, Polk DB. Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial
cells. J Biol Chem 2002;277(52):50959 – 65.
[76] Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16(1):3 – 9.
[77] Hommes DW, Peppelenbosch MP, van Deventer SJH. Mitogen activated protein (MAP) kinase
signal transduction pathways and novel anti-inflammatory targets. Gut 2003;52(1):144 – 51.
[78] Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol
2003;3(12):984 – 93.
[79] Stagg AJ, Hart AL, Knight SC, Kamm MA. The dendritic cell: its role in intestinal inflammation
and relationship with gut bacteria. Gut 2003;52(10):1522 – 9.
[80] Reis e Sousa C. Toll-like receptors and dendritic cells: for whom the bug tolls. Semin Immunol
2004;16(1):27 – 34.
[81] Matsuguchi T, Takagi A, Matsuzaki T, Nagaoka M, Ishikawa K, Yokokura T, et al. Lipoteichoic
acids from Lactobacillus strains elicit strong tumor necrosis factor alpha-inducing activities
in macrophages through Toll-like receptor 2. Clin Diagn Lab Immunol 2003;10(2):259 – 66.
[82] Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli differentially modulate expression of
cytokines and maturation surface markers in murine dendritic cells. J Immunol 2002;168(1):
171 – 8.
[83] Braat H, De Jong EC, Van Den Brande JN, Kapsenberg ML, Peppelenbosch MP, Van Tol EA,
et al. Dichotomy between Lactobacillus rhamnosus and Klebsiella pneumoniae on dendritic
cell phenotype and function. J Mol Med 2004;82(3):197 – 205.
[84] Pessi T, Sqtas Y, Hurme M, Isolauri E. Interleukin-10 generation in atopic children following
oral Lactobacillus rhamnosus GG. Clin Exp Allergy 2000;30(12):1804 – 8.
[85] Miettinen M, Matikainen S, Vuopio-Varkila J, Pirhonen J, Varkila K, Kurimoto M, et al.
Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon
production in human peripheral blood mononuclear cells. Infect Immunol 1998;66(12):6058 – 62.
[86] Hessle C, Hanson LA, Wold AE. Lactobacilli from human gastrointestinal mucosa are
strong stimulators of IL-12 production. Clin Exp Immunol 1999;116(2):276 – 82.
[87] Von der Weid T, Bulliard D, Schiffrin EJ. Induction by a lactic acid bacterium of a population
of CD4+ T cells with low proliferative capacity that produce transforming growth factor b
and interleukin-10. Clin Diag Lab Immunol 2001;8(4):695 – 701.
[88] Veckman V, Miettinen M, Matikainen S, Lande R, Giacomini E, Coccia EM, et al. Lactobacilli
and streptococci induce inflammatory chemokine production in human macrophages that
stimulates Th1 cell chemotaxis. J Leukoc Biol 2003;74(3):395 – 402.
[89] Miettinen M, Lehtonen A, Julkunen I, Matikainen S. Lactobacilli and Streptococci activate
NF-kappa B and STAT signaling pathways in human macrophages. J Immunol 2000;164(7):
3733 – 40.