Total serum immunoglobulin (Ig)E levels are genetically regulated, but the mechanism of inheritance is not well understood. Cytokines produced by T-helper (Th)1 and Th2 lymphocytes control IgE synthesis. Bacterial antigens may favor the development of Th1 cells from naive CD4-positive T cells through a CD14-dependent pathway. CD14 is constitutively expressed on the surface of monocytes and macrophages, and is also present in serum in a soluble form (sCD14). The CD14 gene maps to chromosome 5q31.1, a candidate region for loci regulating total serum IgE. We hypothesized that genetic variants in the CD14 gene could influence Th-cell differentiation and thus total serum IgE. We identified a C-to-T transition at base pair −159 from the major transcription start site (CD14/−159). Among 481 children recruited from a general population sample, frequency of allele C was 51.4%. TT homozygotes had significantly higher sCD14 levels than did carriers of both the CC and CT genotypes (P = 0.01). TT homozygotes also had significantly lower levels of IgE than did carriers of the other two genotypes, but differences were significant only among children who were skin test–positive to local aeroallergens (P = 0.004). There was no association between CD14/−159 and either interleukin (IL)-4 or interferon (IFN)-γ responses by peripheral blood mononuclear cells. However, IFN-γ and IL-4 responses were positively and negatively correlated, respectively, with serum sCD14 levels. We conclude that CD14/−159 plays a significant role in regulating serum sCD14 levels and total serum IgE levels.
Illnesses associated with dysfunction of immunoglobulin (Ig)E responses such as asthma, allergic rhinitis, and atopic dermatitis appear to be on the rise (1), and are presently an important cause of morbidity at all ages (2). These conditions have consistently been found to be associated with increased total serum IgE level in population studies (3). Studies of family aggregation and segregation have suggested that total serum IgE levels are under genetic control (4-12), although the mechanism of inheritance has not been determined. Results of genetic linkage studies have shown that one or more loci present in chromosome 5q may control total serum IgE (11, 13, 14) and may also be involved in the expression of asthma (15, 16) and of bronchial hyperresponsiveness to nonspecific stimuli (17).
Among the genes that have been mapped to chromosome 5q, several have biologic functions that are important in the pathways leading to IgE synthesis: interleukin (IL)-4, IL-5, granulocyte macrophage colony-stimulating factor, IL-9, IL-13, and IL-3. For this reason they have been considered potential candidate genes that may explain the linkage signal observed between total serum IgE and genetic markers in this region. Several of these genes have already been explored for genetic variants associated with total serum IgE and with other IgE-related phenotypes, but the results have not been conclusive (18).
It has recently been suggested that bacterial signals may play a role in promoting T-helper (Th) differentiation and polarization at the time of development of primary immune response (19). In this framework, a potentially important but as yet unexplored gene located in chromosome 5q31.1 is CD14 (20). CD14 is a multifunctional receptor constitutively expressed primarily on the surface of monocytes, macrophages, and neutrophils (mCD14) (21). A soluble form of CD14, sCD14, is abundant in serum and is apparently derived both from secretion of CD14 and from enzymatically cleaved glycosyl-phosphatidylinositol– anchored mCD14 (21, 22). CD14 is a receptor that has specificity for lipopolysaccharides (LPS) and other bacterial wall–derived components (21, 23-25). Engagement of CD14 by these bacterial components is associated with strong IL-12 responses by antigen-presenting cells (23, 24), and IL-12 is regarded as an obligatory signal for the maturation of naive T cells into Th1 cells (26). It is now well established that IgE responses are regulated by inhibitory signals derived from Th1-type cells and by stimulatory signals provided by Th2-type cells (27). Together, these findings led to the hypothesis that variants in the promoter region of the CD14 gene might alter expression of CD14, and this in turn could regulate the proportion of Th2- to Th1-type cells responding to environmental stimuli, thus influencing total serum IgE levels.
Presented here are studies that test this hypothesis. We screened the promoter region of the CD14 gene for potential polymorphisms. We identified one such polymorphism and found that it was associated with total serum IgE levels. The polymorphism was also associated with circulating sCD14 levels, which in turn were correlated with IL-4 and interferon (IFN)-γ responses of peripheral blood mononuclear cells (PBMC).
The subjects of this report were recruited as part of the Tucson Children's Respiratory Study (CRS), a large longitudinal assessment of asthma and allergies in a general population sample enrolled at birth (28). A total of 1,246 healthy newborns and their nuclear families were enrolled between 1980 and 1984. More than three-fourths of these subjects (n = 943) have been followed for the first 14 to 16 yr of life. During an in-depth evaluation performed at a mean age ± SD of 10.8 ± 0.6 yr, the parents of 513 unrelated enrollees who were still living in Tucson gave consent for genetic studies of asthma and allergies. Blood was obtained by venipuncture, and genomic DNA was separated from the cell pellet using conventional methods. Of these 513 subjects, 314 were non-Hispanic whites, 89 were Hispanics, 99 were mixed non-Hispanic whites/other (90% non-Hispanic white/Hispanic), and 11 were of other ethnicities.
The Human Subjects Committee of the University of Arizona (Tucson, AZ) approved this study. Informed consent for genetic studies and for all other procedures was obtained from parents of all subjects involved.
Total serum IgE levels were measured with the paper radioimmunosorbent test using commercially available kits obtained from Pharmacia Diagnostic (Piscataway, NJ). The assay threshold was set at 0.1 IU/ml, as described elsewhere (29).
Skin tests of eight local aeroallergens (Bermuda grass, olive tree, mesquite tree, mulberry tree, careless weed, Alternaria alternata, cat dander, and Dermatophagoides farinae) were performed by the prick technique as described elsewhere (30). Children were considered to be skin test– positive if they showed at least one positive skin test with diameter sums of more than 3 mm.
Serum sCD14 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit supplied by Biosource (Camarillo, CA).
Blood was collected in heparin and processed within 24 h of collection. Blood was layered on Lymphocyte Separation Medium obtained from Organon-Teknika-Cappel (Malvern, PA). The cells at the blood-medium interface were collected, washed with Ca2+- and Mg2+-free Hanks' balanced saline solution, and resuspended in RPMI-1640 medium. Immediately after cell separation was performed, 2 × 106 PBMC were incubated in 1-ml aliquots of RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum, 200 μg/ml l-glutamine, 0.01 M N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a 5% CO2 air atmosphere. Cells were stimulated with 10 μg/ml concanavalin-A (Pharmacia) and 10 ng/ml phorbol myristate acetate (Sigma Chemical Co., St. Louis, MO). Supernatant fluids from these cells and from nonstimulated cells were harvested at 18 to 24 h and stored at −70°C. IFN-γ and IL-4 were assayed in the supernatants using ELISA kits obtained from R&D Systems (Minneapolis, MN).
Genomic DNA from 15 unselected subjects was used to screen for genetic variants in the promoter region of the CD14 gene. Two overlapping polymerase chain reaction (PCR) products based on the reported sequence (31, 32) were amplified. The first product (P1) encompassed the interval between base pair (bp) −513 and bp −61 from the transcription start site, and was amplified using primers 5′-GTGCCAACAGATGAGGTTCAC-3′ and 5′-CCTCTGTGAACCCTGATCAC-3′. The second product (P2), including the interval between bp −151 and bp +291 from the transcription start site, was amplified using primers 5′-CCTGAAACATCCTTCATTGC-3′ and 5′-CGCAGCGGAAATCTTCATC-3′.
Both PCR were carried out in a volume of 25 μl containing 40 ng of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate, 0.8 unit of Taq DNA polymerase supplied by Promega Corp. (Madison, WI), and 5 pmol of each primer. The DNA was denatured at 96°C for 3 min, and temperature cycling was set at 96°C for 40 s, 56°C (P1) or 58°C (P2) for 40 s, and 72°C for 50 s for 38 cycles, followed by a final extension at 72°C for 10 min. The sizes of the generated PCR products were 479 (P1) and 442 bp (P2).
PCR products were electrophoresed on 2% agarose gels and visualized with ethidium bromide staining and ultraviolet illumination. A gel extraction kit obtained from Qiagen Inc. (Valencia, CA) was used to isolate and purify the PCR products, and optical densities of 260 nm (Beckman Instruments, Inc., Fullerton, CA) were used to estimate PCR product concentration. Sequencing reactions were performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer Corp., Foster City, CA): reactions were carried out in a volume of 20 μl containing 4 μl of Terminator Ready Reaction Mix, 50 (P1) or 45 ng (P2) of PCR product, 4 pmol of either forward or reverse primer, and as much ddH2O as needed. Sequencing temperature cycling was 96°C for 30 s, 50°C for 15 s, and 60°C for 4 min for 25 cycles, and 4°C for at least 2 h. After ethanol precipitation, samples were mixed with a loading buffer containing 25 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 50 mg/ml Blue dextran, and deionized formamide in a ratio of 5:1 formamide to EDTA/Blue dextran, heated at 92°C for 3 min, loaded in a 4.75% acrylimide gel, and electrophoresed using an ABI 373 Sequencer (Perkin–Elmer). Data collection and analysis were performed using 373 Data Collection and 373 Sequencing Analysis Software (Perkin–Elmer). Forward- and reverse-complement P1 and P2 sequences were matched with the corresponding reference sequences (31, 32) for each subject using SeqEd software (Perkin– Elmer).
Both parametric and nonparametric analyses of variance were used to assess phenotypic differences between carriers of different genotypes and to assess the association between sCD14 levels and both IFN-γ and IL-4 responses (33). Allele frequencies were estimated as the following sum: (frequency of homozygotes) + 1/2 (frequency of heterozygotes) (34).
The screening of the promoter region of the CD14 gene revealed the presence of only one polymorphism, a C-to-T transition at bp −159 (CD14/−159) from the major transcription start site (Figure 1). To genotype a large number of subjects for CD14/−159, a restriction fragment assay was developed. A 497-bp PCR product encompassing the interval bp −513/bp −17 was generated using primers 5′-GTGCCAACAGATGAGGTTCAC-3′ and 5′-GCCTCTGACAGTTTATGTAATC-3′.
Commercially available AVAII endonuclease (New England Biolabs, Beverly, MA) is specific for the sequence GGTCC, which is present in this PCR product only among carriers of the CD14/−159 T allele. The assay thus yields one 497-bp band for the CC genotype (Figure 2, lane 4); three bands of 144, 353, and 497 bp for CT heterozygotes (lanes 1, 2, 5–8, 10, and 11); and two bands of 144 and 353 bp for TT homozygotes (lanes 3 and 9). Genotypes were obtained for 481 CRS enrollees. Frequencies of CC, CT, and TT genotypes were 29.4, 49.4, and 21.3%, respectively. Allele frequency for C was estimated at 51.4%, and distribution was very similar to the expected Hardy– Weinberg equilibrium. There were no significant ethnic differences in allele frequency among the 470 subjects who were Hispanic, non-Hispanic white, or mixed (see Materials and Methods).
We assessed serum levels of sCD14 in 67 unselected CC subjects and 42 unselected TT subjects (with the performer of the assays blinded as to genotype). Median (interquartile distance) values were 4.1 μg/ml (3.6 to 4.5) for CC subjects and 4.5 μg/ml (4.1 to 5.0) for TT subjects (P = 0.01) (Figure 3).
Among non-Hispanic whites, those with the TT genotype had lower total serum IgE levels than both CC homozygotes and heterozygotes. When stratified by positive and negative allergen skin-test reactivity, differences were statistically significant only among those positive for allergen skin tests (Table 1). Heterozygotes had very similar total serum IgE levels when compared with CC homozygotes, suggesting a dominance of the C allele. Among skin test– positive subjects, the combined CC plus CT group (n = 124) had geometric mean total serum IgE of 178.4 IU/ml (confidence interval [CI], 137.4 to 231.9), which was significantly higher than that of TT homozygotes (geometric mean, 81.4; CI, 51.6 to 128.3; P = 0.004).
|CD14/−159Genotypes||n||Total Serum IgE among Skin Test–Negative Children||n||Total Serum IgE among Skin Test–Positive Children|
|P = 0.735||P = 0.016|
There was no association between CD14/−159 genotypes and prevalence of skin-test reactivity to aeroallergens in either Hispanic or non-Hispanic white children (data not shown). However, among skin test–positive non-Hispanic white children, the number of positive skin tests was strongly associated with the CD14/−159 genotypes (Table 2). Again, CC homozygotes had mean numbers of positive skin tests that were very similar to those of CT heterozygotes, and the difference in number of positive skin tests between the combined group (CC + CT) and that of TT subjects was highly significant (P = 0.001).
|CD14/−159Genotypes||n||Mean Number of Positive Skin Tests|
|P = 0.0063|
Among Hispanic children there was no significant association between total serum IgE levels and CD14/−159 genotypes for either skin test–positive or skin test–negative children (Table 3). There was also no association between number of positive skin tests and CD14/−159 genotypes among skin test–positive Hispanic children (data not shown).
|CD14/−159Genotypes||n||Total Serum IgE among Skin Test–Negative Children||n||Total Serum IgE among Skin Test–Positive Children|
|P = 0.38||P = 0.39|
We assessed IFN-γ and IL-4 responses to concanavalin-A and phorbol myristate acetate stimulation of PBMC in children assessed for sCD14 levels. The proportion of children with detectable IL-4 responses did not differ by genotype (19.0% in 63 CC subjects versus 20.0% in 40 TT subjects, P = 0.9). Geometric mean IFN-γ responses (95% CI) were 63.6 pg/ml (49.2 to 82.2) for 67 CC subjects and 67.8 pg/ml (50.9 to 90.5) for 41 TT subjects (P = 0.7).
Figure 4 shows the results of a multiple regression in which sCD14 was the dependent variable and both IFN-γ (in four categories) and IL-4 (as detectable versus undetectable) were introduced into the model as independent variables. Subjects with low IL-4 responses had significantly higher circulating levels of sCD14 when compared with subjects with high IL-4 responses. Subjects with high IFN-γ responses had higher levels of sCD14 than did subjects with low responses.
We have identified a polymorphism in the 5′ flanking region of the CD14 gene at position −159 from the transcription start site. This polymorphism is very frequent among both Hispanic and non-Hispanic white subjects, with approximately half of all chromosomes carrying the T allele and half the C allele. Although the specific role of this polymorphism in the CD14 gene transcription has not yet been elucidated, it appears that the T allele increases CD14 expression, as assessed by the increased sCD14 levels in homozygous carriers of this allele. It is also possible, however, that CD14/−159 may be in linkage disequilibrium with a different polymorphism located in this region that controls the CD14 gene transcription rates. Functional evaluation of the role of CD14/−159 in determining transcription rates of the CD14 gene will thus be necessary to clarify this issue.
CD14/−159 TT homozygotes showed significantly lower levels of total serum IgE than did both CC and CT subjects. The mechanisms that may explain the association between CD14/−159 and total serum IgE are not yet well understood. IgE responses are regulated by the cytokines produced by Th1-like and Th2-like cells (27). IL-4, produced by Th2-like cells, is one of two major signals determining the ɛ class switch in B-cell IgE synthesis (35). Both production of IgE by B cells and differentiation of Th2-like cells (35) are inhibited by IFN-γ, which is produced by Th1-like cells. There is increasing evidence suggesting that the predominant type of response (Th1- or Th2-like) to a given antigen is determined at the time of the primary encounter with the antigen (36, 37). Our data demonstrate an association between sCD14 levels and low IL-4 and high IFN-γ, suggesting a role for CD14 in influencing T-cell differentiation. Thus, CD14/−159 may influence sCD14 levels (and presumably mCD14 expression), which in turn may regulate IgE responses and consequently serum IgE levels.
It has been suggested that dendritic cells (DCs) are professional antigen-presenting cells that play a major role in primary responses to antigens and in determining differentiation of naive Th cells into either Th1- or Th2-like cells (19). In the absence of IL-12–inducing factors, DCs have been shown to induce both IL-4 and IFN-γ production in maturing naive Th cells (38), supporting the hypothesis that IL-12 production by DCs may be an obligatory signal for the differentiation of naive T cells into Th1-like cells (26). Certain adjuvants have been found to enhance or induce IL-12 secretion, including the engagement of CD14 by LPS and other bacterial cell wall–derived molecules (21, 23-26, 39). CD14 may thus serve as a crucial link between nonadaptive and adaptive responses to environmental antigens (40). Interestingly, DCs do not express mCD14 on their surfaces (41, 42), but can mount IL-12 responses to LPS stimulation by recognizing complexes formed as a result of interactions among sCD14 and LPS (40). Recent evidence suggesting that cytokine responses by DCs to LPS show dose-response relations with sCD14 concentration support the potential involvement of these complexes in DC activation (40). LPS is ubiquitous in nature, being present in normal indoor environments as a constituent of house dust (43), and hence is ideally localized to play a “bystander” role in immune responses to inhalant allergens that are controlled by the DC network in the airway epithelium (44, 45). LPS derived from respiratory bacterial infections may play a similar role. In addition, microbial stimulation via LPS may exert more generalized effects on immune function, which influence Th1/Th2 switch regulation in allergen-specific immune responses. In particular, recent studies suggest that key events determining the cytokine phenotype of long term Th-cell memory responses to allergens occur during infancy and early childhood, a life period during which the Th2-polarization characteristic of the fetal immune system is progressively replaced by Th1 dominance (38, 46). The primary trigger for postnatal maturation of Th1 function is believed to be microbial stimulation, in particular via bacterial LPS derived from the commensal flora of the gastrointestinal tract (47, 48). Consequently, any alteration in CD14 function may interrupt this form of signaling through to the adaptive immune system, and this may contribute to the transient developmental defect in Th1 function now recognized as a characteristic of the atopic phenotype.
Experimental support has recently been provided for this hypothesis. Yoshimoto and coworkers (49) reported that SJL mice, a strain known to be poor producers of IgE, showed no intrinsic deficits in B-cell IgE responses when directly stimulated with LPS and IL-4. However, macrophages from SJL mice, when stimulated with LPS, produced increased amounts of both IL-12 and IL-18, and these two cytokines induced T cells to produce increased amounts of IFN-γ. As a consequence, proliferation and IgE production by LPS and IL-4 stimulated B cells were significantly reduced. Furthermore, the ability of SJL B cells to produce normal amounts of IgE was restored either by addition of anti–IL-12 plus anti–IL-18 antibodies or by removal of macrophages (49). These results strongly suggest that a mechanism that increases macrophage susceptibility to LPS stimulation may increase macrophage production of cytokines that inhibit a Th2-predominant immunity.
Further support for this hypothesis is our finding that sCD14 levels were directly correlated with IFN-γ responses and inversely correlated with IL-4 responses by mitogen-stimulated PBMC. The preponderance of Th2-like cytokines such as IL-4 may thus tilt the immune response in favor of IgE production, and may explain the higher levels of IgE observed in CC subjects, who had lower levels of circulating sCD14.
Among TT homozygotes, lower levels of IgE were observed in both skin test–positive and –negative subjects, but results only reached statistical significance among the latter group (see Table 1). Skin test–negative subjects had markedly lower geometric mean serum IgE levels than did skin test–positive subjects, suggesting that the main factor determining IgE production in this population involves the susceptibility to becoming sensitized to environmental aeroallergens. Because susceptibility to becoming sensitized to at least one aeroallergen was independent of CD14/−159 genotypes, other genes may modulate such susceptibility, thus interacting with CD14/−159 in determining total serum IgE levels. It is possible that in the absence of such epistatic influences, the effect of CD14/−159 on total serum IgE levels would be more difficult to detect, as suggested by our results among skin test–negative subjects. Interestingly, we found that among skin test–positive subjects, the number of positive skin-test reactions was strongly associated with CD14/−159 genotypes, with the TT genotype having a significantly lower mean number of positive skin tests when compared with CC and CT genotypes. These results suggest that, although the likelihood of becoming sensitized to any aeroallergen is independent of CD14/−159 genotypes, the propensity to mount IgE responses to different aeroallergens that are sufficiently strong to be detected by skin-prick test does depend upon CD14/−159 genotypes. In population studies, prevalence of asthma is a function both of total serum IgE levels (50) and of the number of positive skin tests to local aeroallergens (3). It is tempting to speculate that linkage signals between asthma and markers in chromosome 5q may be due, at least in part, to the association between CD14/−159 and both total serum IgE levels and the number of positive skin tests to aeroallergens. Unfortunately, our general population study included too few asthmatic subjects to allow us to test this hypothesis.
We found that the association between total serum IgE levels and CD14/−159 genotypes reached statistical significance only among non-Hispanic white children. This is in apparent agreement with recent reports from the Collaborative Study of the Genetics of Asthma, which indicate that linkage between markers in chromosome 5q and asthma is present among non-Hispanic whites but not among Hispanic subjects (51). In addition, Laitinen and colleagues (52) were unable to show linkage between total serum IgE levels and markers in chromosome 5q in a relatively isolated population in Finland. Thus, there may be a significant ethnic heterogeneity in the genetic determination of total serum IgE levels. Our results should be interpreted cautiously, however, because the number of Hispanic subjects included may have been too small to provide sufficient power in our sample.
The identification of a genetic variant associated with sCD14 levels may have implications that go beyond those discussed herein related to allergic conditions. Apart from its role in primary immune responses, CD14 plays a crucial role as a mediator of inflammatory responses during gram-negative sepsis (53). Clinical studies have suggested that levels of sCD14 measured shortly after a diagnosis of gram-negative sepsis are strongly associated with subsequent mortality (54). Overexpression of the CD14 gene in transgenic mice is associated with increased mortality after intravenous administration of LPS (55). CD14-deficient mice produced by gene targeting in embryonic stem cells, on the other hand, show markedly lower mortality rates after LPS administration when compared with their wild-type counterparts (56). It is thus tempting to speculate that, although TT subjects may be at decreased risk of developing high IgE responses to environmental stimuli, they may be at increased risk of mortality associated with gram-negative sepsis.
In summary, we found that a genetic polymorphism in the 5′ regulatory region of the CD14 gene may explain, at least in part, the linkage signal observed between total serum IgE levels and markers in chromosome 5q. We demonstrated that CD14/−159 influences serum sCD14 levels. Further elucidation of the molecular pathways that explain this association may provide important new insights into the mechanisms that regulate IgE responses in humans.
The authors thank Debra A. Stern, Susan Solomon, and Penelope E. Graves for their invaluable technical support; and Maureen Driscoll for preparing the manuscript. This study was supported by grants HL14136 and HL56177 from the National Heart, Lung and Blood Institute (NHLBI). One author (F.D.M.) was also funded by a Research Development Award for Minority Faculty (HL03154) from NHLBI.
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* Genbank Accession No. AFO97335.
Abbreviations: base pair(s), bp; polymorphic locus at bp −159 from the AGT transcription start site of the CD14 gene, CD14/−159; dendritic cell, DC; interferon, IFN; immunoglobulin, Ig; interleukin, IL; lipopolysaccharide(s), LPS; a multifunctional receptor constitutively expressed primarily on the surface of monocytes, macrophages, and neutrophils, mCD14; peripheral blood mononuclear cells, PBMC; polymerase chain reaction, PCR; a soluble form of CD14, sCD14; T-helper, Th.