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Published ahead of print on January 24, 2008, doi:10.1165/rcmb.2007-0143OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 733-737, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0143OC

Correlation of Th1-Type Cytokine Expression and Induced Proliferation to Lipopolysaccharide

Michael R. Goldberg1,2, Orna Nadiv1, Noemi Luknar-Gabor1, Galia Zadik-Mnuhin2, Joseph Tovbin3 and Yitzhak Katz1,2

1 Institute of Allergy and Immunology, 2 Department of Pediatrics, and 3 Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin, Israel

Correspondence and requests for reprints should be addressed to Michael R. Goldberg, M.D., Ph.D., Institute of Allergy and Immunology, Assaf Harofeh Medical Center, Zerifin, 70300, Israel. E-mail: goldbergsm{at}yahoo.com


   Abstract
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 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Recent evidence from mouse models indicates that neonatal exposure to lipopolysaccharide (LPS) can prevent experimentally induced allergic disease. Furthermore, we noted that human cord blood mononuclear cells (CBMC) have an increased proliferative response to LPS relative to their respective maternal peripheral blood mononuclear cells (PBMC). We sought, therefore, to examine the cytokine expression profile induced by LPS in CBMC and its relationship to the LPS-mediated proliferative response. CBMC and maternal PBMC were evaluated for IL-10, IL-4, IL-13, IL-12{alpha}, and IFN-{gamma} expression after LPS stimulation by real-time PCR. IFN-{gamma} secretion was detected by enzyme-linked immunosorbent assay. LPS increased IFN-{gamma} and IL-13, but decreased IL-4 expression in CBMC (P < 0.024, P < 0.014, and P < 0.027, respectively). In PBMC, however, no significant changes in expression were noted after LPS stimulation. Stimulation by LPS significantly increased the secretion of IFN-{gamma} in CBMC compared with PBMC at the two concentrations analyzed (1 ng/ml, P < 0.048; 10,000 ng/ml, P < 0.003). The magnitude of the LPS-mediated proliferative effect in CBMC directly correlated to the level of induction of IFN-{gamma} (P < 0.01), but inversely correlated to the induced levels of IL-4 and IL-13 (P < 0.01 and P = 0.01, respectively). No association of the CBMC proliferative response to IL-12{alpha} or IL-10 was noted. Thus, a high proliferative response to LPS in CBMC correlates with a change from a Th2- to Th1-induced cytokine expression profile. Since early exposure to LPS may protect against allergic disease, one may speculate that an aberrant response to LPS may increase the likelihood of developing overt disease in susceptible individuals.

Key Words: asthma hygiene hypothesis • chemokines • innate immunity



   CLINICAL RELEVANCE
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 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
These results describe for the first time a mechanism by which human cord blood mononuclear cells may be influenced by the innate immune system to mitigate against Th2-mediated disease.

 
Over the past several decades there has been a paradoxical rise in the incidence of both atopic and autoimmune diseases, suggesting that the change to a more hygienic lifestyle may have impact on the regulatory functions of the immune system (1, 2). Epidemiologists had previously described a protective effect of exposure to stables and farm milk early in life, particularly if the exposure occurred before one year of age (3). Stemming from these findings, several investigators raised the possibility that exposure to higher levels of endotoxin (lipopolysaccharide, LPS) may be providing the protection. In one large-scale multicenter study, endotoxin levels in samples of dust from children's mattresses were inversely correlated to the occurrence of hay fever, atopic asthma, and atopic sensitization (4).

In addition to the epidemiological evidence, experimental studies in the mouse suggest that neonatal exposure to LPS can prevent Th2-mediated allergic disease. As an example of such a mechanism, Toll-like receptor-4–dependent signals provided by the intestinal commensal flora inhibit the development of Th2-mediated allergic responses to food antigens (5). Diminished experimental allergic airways disease has also been reported after exposure to LPS. In one report, intranasal application of LPS to newborn mice and subsequent sensitization to, and challenge with, ovalbumin directed mucosal responses toward tolerance. Abrogation of airway hyperresponsiveness, a significant decrease in IL-13 from bronchoalveolar lavage fluid, and a decrease in specific immunoglobulin (Ig)E were also noted (6). Interestingly, even prenatal exposure of mothers during pregnancy prevented allergic sensitization and airway inflammation (7). It should be noted, however, that airway hyperresponsiveness could only be diminished when combined with further postnatal LPS exposure (8).

The above experimental evidence clearly indicates that neonatal exposure to LPS can prevent Th2-mediated allergic disease. The mechanisms by which this occurs, however, and its applicability to humans, has not been explored. We therefore began our research aiming to fundamentally characterize the neonatal cells' response to LPS, with LPS serving as a surrogate activator of the innate system. We hypothesized that the neonate harbors a population of cells that identify and react to LPS. In accordance with this, we previously demonstrated that cord blood–derived mononuclear cells (CBMC) proliferate in response to LPS (9). Furthermore, LPS synergized with immobilized anti-human CD3{varepsilon} mAb to augment the proliferative response in CBMC, but failed to do so in maternally derived peripheral blood mononuclear cells (PBMC) (9). In this article we explore the effects of LPS on cytokine expression and the relationship between cytokine expression and the proliferative effects induced by LPS.


   MATERIALS AND METHODS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Ficoll-Paque PLUS and tritiated thymidine were bought from Amersham Biosciences (Uppsala, Sweden). Phytohemaglutinnin (PHA), LPS from Escherichia coli serotype 0111:B4, Tween 20, avidin peroxidase conjugate, ABTS Liquid Substrate Solution, and polymyxin B sulfate were purchased from Sigma (St. Louis, MO). Bovine serum albumin was obtained from USB (Cleveland, OH). RPMI, fetal calf serum (FCS), L-Glutamine, Hanks' buffered saline solution, HEPES, and Dulbecco's PBS were purchased from Biological Industries Ltd. (Beit Haemek, Israel).

Isolation of Blood Mononuclear Cells via Density Gradient Centrifugation
The Institutional Review Board at the Medical Center approved the study protocol according to the Helsinki Declaration, and written consent was obtained from the donors. Twenty-eight CBMC were analyzed for their response to LPS (10 µg ml–1), including 15 in which their respective maternally derived PBMC response was compared. In addition, 14 CBMC were analyzed for their response to LPS at 1 ng ml–1. All infants were full-term (>38 wk). Only routine uncomplicated pregnancies were included in our analysis. Diabetics on insulin, patients with preeclampsia, or patients with fever suggesting of chorioamnionitis and any other high-risk pregnancies were excluded. Eighteen neonates were born through elective cesarean section, before labor, while 10 were born through a vaginal delivery. Blood was drawn from the mother upon placement of the intravenous line. Cord blood was obtained from catheterization of the umbilical vein with an 18-gauge syringe and placed into vacutainers containing EDTA. CBMC and PBMC were isolated after a Ficoll-Paque centrifugation as previously described (9). After erythrocyte lysis in the neonate (9), fluorescence-activated cell sorter analysis of CBMC and PBMC revealed that our starting populations contained 39% (n = 7) and 54% CD3+ cells, respectively (n = 5).

Cytokine Expression Profile
Isolated PBMC and CBMC were seeded at 1.5 to 3.0 x 106 cells/well of a 24-well plate in RPMI containing 5% heat-inactivated FCS. Total mRNA was isolated from CBMC after culture in LPS for 72 hours using TRI Reagent (Sigma, Rehovot, Israel), according to the manufacturer's instructions. Our choice of 72 hours was based on our initial experiments demonstrating the optimal proliferative response in CBMC at this time. Reverse transcription was performed using the Reverse-iT first-strand Synthesis Kit (ABgene, Surrey, UK). Amplification of IL-4, IL-13, IFN-{gamma}, IL-10, IL-12{alpha}, and GAPDH cDNA by real-time PCR used LightCycler FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). The housekeeping gene GAPDH was used as an internal control for normalization of mRNA levels. The primers pairs used and resulting size transcript produced are listed in Table 1. A stimulation index was determined as the relative amounts of specific mRNA generated for each cytokine after LPS treatment compared with cells in medium alone. Data correlating the proliferative response to LPS (10 µg/ml) to the corresponding induced expression of cytokines in CBMC used the proliferative data previously described (9), in which CBMC were seeded at 2 x 105 cells/well.


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  		TABLE 1. PRIMERS USED FOR REAL-TIME PCR

 
Measurement of IFN-{gamma}
The human IFN-{gamma} enzyme-linked immunosorbent assay (ELISA) Development Kit and standards were purchased from Cytolabs (Rehovot, Israel). The ELISA was performed as per the manufacturer's guidelines. Briefly, 400 nanograms of anti-human IFN-{gamma} mAb was incubated overnight at 4°C in 96-well ELISA microplates bought from Nunc (Roskilde, Denmark). Wells were then washed with PBS containing 0.05% Tween-20 and subsequently blocked with 1% (wt/vol) bovine serum albumin for 2 hours at room temperature. After blocking, supernatants were added at 2-fold dilutions in the presence of the detection antibody (biotinylated anti-human IFN-{gamma} mAb) for 2 hours. Excess antibody was then washed away and wells were incubated with avidin peroxidase (Sigma, St. Louis, MO). After 30 minutes, wells were washed and incubated with the reaction substrate, ABTS, for 20 minutes. The optical density was measured using a Dynatech MR5000 Elisa reader at 405 nm. Quantitation of the amount of IFN-{gamma} secreted in the supernatant was derived from a standard curve (80–1,600 pg/ml) performed in each experiment. Four wells per condition were assayed. Results presented are the cumulative data of 16 maternal-neonate pairs.

Statistics
A paired t test was used for the comparison of PBMC and CBMC baseline expression of cytokine expression, for the CBMC response to LPS, and for the evaluation of LPS stimulation of IFN-{gamma} secretion in CBMC relative to their respective maternally derived PBMC values. An unusually high CBMC IFN-{gamma} response to LPS at 1 ng ml–1 (>400-fold increase) was not included in the statistical analysis. The Pearson's correlation coefficient was used to determine the degree of association between the proliferative responses previously described (9) to the levels of induced cytokine expression. The statistical significance of the correlation was determined by a paired t test. A linear regression analysis between the two variables was performed and the best fit curve drawn on the data points. For IFN-{gamma} and IFN-{gamma}/IL-4, expression values were plotted on a logarithmic scale, and an exponential curve was best fit to the proliferative data.


   RESULTS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Regulation of Cytokine Expression in CBMC by LPS
If exposure to LPS is protective against a predisposition of Th2-mediated responses, as some epidemiological studies suggest (4), we hypothesized that a possible mechanism for this finding may be through altering the balance of expression between Th1- and Th2-type cytokines. Basal expression analysis by real-time PCR was first performed for IL-10, IL-4, IL-12, IFN-{gamma}, and IL-13 and the results were normalized to GAPDH. A significant increase in basal IL-10 expression was noted in the PBMC versus CBMC (Figure 1, P < 0.009). No significant differences were noted, however, for IL-4, IL-12, IFN-{gamma}, or IL-13. The response to LPS was then evaluated. No significant differences were noted in PBMC after LPS treatment (data not shown). Responses to concentrations of LPS at 1 ng/ml and 10 µg/ml were similar; however, a more pronounced effect was noted at the higher dose (Figure 2). LPS significantly increased the expression of IFN-{gamma} and IL-13 in CBMC (LPS 1 ng ml–1, P < 0.04; LPS 10 µg ml–1, P < 0.024; and LPS 1 ng ml–1, P < 0.033; LPS 10 µg ml–1, P < 0.014, respectively) but decreased the expression of IL-4 by 37% (LPS 10 µg ml–1, P < 0.027) (Figure 2). A decrease in IL-12{alpha} mRNA expression after LPS treatment was also noted (LPS 10 µg ml–1, P < 0.052), albeit where we determined by ELISA, LPS (10 µg/ml) induced abundant IL-12 p70 (mean >2,000 pg/ml, n = 3, data not shown). When the induction of cytokine expression (LPS 10 µg ml–1) was compared between PBMC and CBMC, the latter expressed greater amounts of IFN-{gamma}, IL-13, and IL-10 (P < 0.018, P < 0.002, and P < 0.048, respectively) (Figure 3).


Figure 1
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  		Figure 1. Basal cytokine expression in cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC). Real-time PCR analysis was performed for IL-10, IL-4, IL-12{alpha}, IFN-{gamma}, and IL-13, in matched maternally derived PBMC and their respective CBMC. The data are derived from at least 14 matched pairs, except for IL-12{alpha}, for which 10 pairs were analyzed. Bars are presented with ± SE (*P < 0.009).

 

Figure 2
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  		Figure 2. Regulation of cytokine expression by lipopolysaccharide (LPS) in CBMC. Presented are the induced cytokine mRNA expression levels in CBMC after LPS stimulation at 1 ng/ml ([A]) and 10 µg/ml ([B]), divided by their respective control expression. mRNA expression was normalized to GAPDH in each sample. Each individual circle at 10 µg/ml represents a single recorded response, while at 1 ng/ml each circle represents the average CBMC response performed in duplicate. The average for the group is represented by a bar in the figure ± SE. Statistically significant increases were noted in CBMC for IFN-{gamma} (LPS 1 ng ml–1, P < 0.04; LPS 10 µg ml–1, P < 0.024) and IL-13 (LPS 1 ng ml–1, P < 0.033, LPS 10 µg ml–1, P < 0.014). A statistically significant decrease in IL-4 expression was noted after LPS treatment (LPS 10 µg ml–1, P < 0.027, n = 28).

 

Figure 3
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  		Figure 3. Comparison of the cytokine expression profile in PBMC and CBMC in response to LPS. LPS stimulation (10 µg/ml) in matched PBMC–CBMC pairs were evaluated for the expression of IL-10, IL-4, IL-12{alpha}, IFN-{gamma}, and IL-13 by real-time PCR and normalized for the expression of GAPDH ± SE (bars). The data are derived from at least 13 matched pairs, except for IL-12{alpha}, for which 11 pairs were analyzed (*P < 0.018, **P < 0.048, ***P < 0.002).

 
In order to correlate the expression of IFN-{gamma} on the mRNA and protein levels, we quantitated by ELISA the amount of IFN-{gamma} secretion after the stimulation of CBMC and PBMC for 72 hours (Figure 4). CBMC from neonates secreted little IFN-{gamma} in response to PHA, in contrast to the robust response of maternal cells. This result has been previously reported (10). In response to LPS, CBMC secreted significantly greater amounts of IFN-{gamma} than PBMC at both 1 ng/ml (P < 0.048) and 10 µg/ml (P < 0.003). Thus, CBMC were shown to be potent secretors of IFN-{gamma} when exposed to LPS. This is in contrast to the minimal CBMC response when triggered by exposure to specific environmental allergens (11), activation by crosslinking via anti-CD3 antibodies (12), or global T cell activators such as Con A and PMA (13).


Figure 4
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  		Figure 4. Quantitation of IFN-{gamma} secretion in PBMC and CBMC pairs by enzyme-linked immunosorbent assay (ELISA). Supernatants of PBMC and CBMC cultured for 3 days in medium, phytohemaglutinnin (PHA), or LPS were measured for IFN-{gamma} secretion by ELISA. Presented are the mean (in pg/ml) ± SE from 16 maternal-infant pairs, which reveal that PBMC as compared to CBMC produce greater amounts of IFN-{gamma} in response to PHA (P < 0.0007), while the reverse is true for their respective responses to LPS (1 ng/ml, P < 0.048; 10 µg/ml, P < 0.003).

 
Relationship between Cytokine Expression and Proliferative Response to LPS
We and others have reported that the overwhelming majority of CBMC proliferate in response to LPS (9, 14), in contrast to the maternal proliferative response. Therefore, we next addressed whether there existed a correlation between the previously reported proliferative responses noted in CBMC (9), and the cytokine expression profile induced by the same LPS treatment. In initial experiments no correlation was noted between the constitutive expression levels of IL-4, IL-12, IL-13, IL-10, and IFN-{gamma} and CBMC's proliferative response to LPS (data not shown). We next compared the cytokine expression from CBMC in response to LPS (10 µg/ml) relative to its proliferative response to LPS (10 µg/ml). Figures 5A and 5B demonstrate that an inverse correlation exists between proliferation mediated by LPS and the expression of IL-4 and IL-13. In contrast, the greater the magnitude of the proliferative response to LPS, the more IFN-{gamma} is expressed (Figure 5C). Similarly, a direct correlation was noted between the proliferative response to LPS and the ratio of induced IFN-{gamma}/IL-4 expression (Figure 5D). No correlations were noted between induced IL-10, IL-12{alpha}, and the LPS-mediated proliferation (Figures 5E and 5F). Thus, the greater the proliferative response of CBMC to LPS, the greater was the likelihood for a shift from the expression of the Th2 cytokines, IL-4 and IL-13, and to the generation of IFN-{gamma}.


Figure 5
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  		Figure 5. Relationship between cytokine expression and proliferative response to LPS. LPS (10 µg/ml) induced mRNA expression (LPS/C) in CBMC of IL-10, IL-4, IL-12{alpha}, IFN-{gamma}, and IL-13 was plotted against their respective proliferative index (LPS/C). mRNA expression was quantitated by real-time PCR and normalized to GAPDH. (A and B) There was a significant inverse correlation between the ability to express IL-4 and IL-13 in response to LPS and the cell's proliferative response to LPS (r = 0.6979, P < 0.01 and r = 0.59, P = 0.01, respectively). (C and D) There was a significant correlation between induced expression of IFN-{gamma} and the IFN-{gamma}/IL-4 ratio and the cell's proliferative response to LPS (r = 0.647, P < 0.01 and r = 0.746, P = 0.01, respectively). (E and F) No correlation was noted between the levels of induced IL-10 or IL-12{alpha} and the proliferative response.

 

   DISCUSSION
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
These studies describe, for the first time, a correlation between LPS-mediated proliferation in CBMC and a change from a Th2 to Th1 cytokine expression profile. We previously noted that 88% of CBMC proliferated in response to LPS (10 µg/ml), in contrast to only 31% of their respective maternally derived PBMC (9). In this study we observe that the greater the proliferative effect to LPS, the lower the expression of IL-4 and IL-13 by CBMC. Furthermore, CBMC with strong proliferative responses to LPS, expressed greater amounts of IFN-{gamma}. Prior evidence derived from the cytokine expression profile after exposure to environmental allergens implicated a Th2 skewing in the neonate (11). Our results would suggest that a heightened proliferative response to LPS may be one mechanism by which to balance the cytokine expression profile in the neonate. It remains to be determined whether the change in cytokine expression is a direct result of a new subset of responding T cells, a secondary effect mediated by a different class of cells, or a result of the generation of a new mediator.

While the greater proliferative response to LPS correlated to an increase in IFN-{gamma} production, no correlation was noted to the expression of IL-12(p35). Earlier reports have demonstrated a profound defect in IL-12p70 secretion in the CBMC response to LPS (15). Both a 3-fold lower half-life of IL-12 p40 mRNA in activated CBMC compared with activated PBMC (16), and a defect in IL-12(p35) synthesis in dendritic cells derived from adherent CBMC, were noted (12). A defect in nucleosome (nuc-2) remodeling preventing IL-12(p35) gene transcription was demonstrated in these cord blood–derived dendritic cells (17). Whether a similar defect is occuring in CBMC is unclear, although a decrease in IL-12(p35) gene transcription in response to LPS was noted here (P < 0.052). Alternatively, the increased IFN-{gamma} expression may lead to a subsequent down-regulation of IL-12 expression in CBMC. Importantly, the defect in nucleosome remodeling, as well as IL-12(p35) gene transcription, were restored upon addition of recombinant IFN-{gamma}. A feedback loop mediated by IFN-{gamma} in response to LPS may therefore mitigate the decrease in expression of IL-12 in the neonate.

We noted an increase in basal expression of IL-10 in maternally derived PBMC compared with CBMC. An abundance of IL-10 in gestational tissues was previously identified as a critical determinant of resistance to preterm labor in mice (18). In addition, given the role proposed for T regulatory cells and their generation of IL-10 to maintain fetal tolerance (19), it will be of interest, therefore, to compare the IL-10 levels from these women after their pregnancy-induced changes are terminated. There was a statistically significant increase of IL-10 expression after LPS stimulation in CBMC compared with PBMC. This is consistent with other reports demonstrating increased secretion of IL-10 in CBMC after LPS treatment (15).

Interestingly, dendritic cell IL-10 production in response to LPS is diminished in children with asthma (20), consistent with the hypothesis that IL-10 may act to regulate both Th2- and Th1-mediated disease (21).

The response to LPS may very well depend on the route of administration, dosing, and age of exposure. When administered by aerosol in normal adult subjects, it can cause bronchoconstriction and pulmonary inflammation (22). However, when LPS exposure occurs at a young age, both experimental and epidemiological evidence points to a protective role against Th2-mediated disease. One mechanism may be through the LPS-induced proliferation that correlated with a decrease in IL-13–induced expression, as demonstrated here.

The proliferative response to LPS in CBMC may be an important early determinant of an individual's susceptibility to atopy. Future studies will determine whether the degree of the proliferative response to LPS correlates to the atopic status of the child. It is intriguing to speculate that individuals susceptible to atopic diseases require in their early years a greater cumulative exposure to LPS, or exposure to additional factors, to be afforded protection against Th2-mediated disease.


   Acknowledgments
 
The authors are indebted to Pnina Koch for expert technical assistance. They also acknowledge Ora Soffrin and Amalya Dror for their help in obtaining blood samples for this study.


   Footnotes
 
This research was supported by Spunk Foundation International.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0143OC on January 24, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 23, 2007

Accepted in final form October 17, 2007


   References
Top
 Abstract
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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