Genetics and specific immune response in allergy to birch pollen and food: evidence of a strong, positive association between atopy and the HLA class II allele HLA-DR7.

BACKGROUND: In some geographic areas birch pollen represents the most prominent cause for airborne allergic diseases. Up to 70% of patients allergic to birch pollen are hypersensitive to fruits, especially apples. Associations have been found, in some instances, with a sensitivity to aeroallergens and HLA class II genes. OBJECTIVES: We investigated whether susceptibility or resistance to birch pollen allergy with and without food allergy was associated with HLA class II genes. METHODS: Blood samples were obtained from 2 groups of unrelated European-born white adults: 42 atopic patients (31 of them with asthma) and 42 healthy control subjects with no personal or familial history of asthma or atopy. Their antibody responses to birch pollen, apples, grass, and weed pollens were evaluated by skin tests, RASTs, and immunoprints. Genomic DNA was extracted from PBLs. The exons of DQA1, DQB1, DRB1, and DPB1 genes were selectively amplified by using the PCR method. Genotyping was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which recognize allelic variations in the polymorphic exon. RESULTS: We found no significant differences in the frequency of DPB1 alleles between patients and control subjects. HLA class II DR4 and/or DR7 alleles were present in 42.6% of the patients and in only 2.4% of the healthy subjects. These results confirm a previous study of a group of polysensitized atopic patients, which showed that DR4 and DR7 alleles were rare in healthy control subjects and frequently observed in atopic subjects with or without concomitant asthma. CONCLUSION: We conclude that the allele HLA-DR7 is significantly involved in the presentation of apple and pollen allergens. However, we suggest that this susceptibility is more related to atopy than to specific responses to allergens.

Analysis of hsp70 gene polymorphism in allergic asthma.

BACKGROUND: Allergic asthma is a multifactorial and probably multigenic inflammatory disease of the upper airways, and has been associated with the HLA class II alleles DR4 and DR7. Here we investigated possible associations with other polymorphic susceptibility/resistance genes located within the major histocompatibility complex, i.e., the genes coding the major 70-kDa heat-shock proteins (HSP; Hsp70) hsp70-1, hsp70-2, and hsp70-HOM, whose products are overexpressed in the bronchi of asthmatic patients. METHODS: Genomic DNA was extracted from peripheral blood lymphocytes or buccal epithelial cells of 48 patients with allergic asthma and 31 selected nonatopic control subjects, in whom we previously reported a strong association of atopy with DR4/DR7 alleles. RESULTS: No evidence was found for an independent role of hsp70 gene polymorphism in susceptibility to allergic asthma. However, hsp70 alleles might be involved in extended haplotypes of HLA markers. CONCLUSIONS: Our data suggest that Hsp70 overexpression in asthma results from complex interactions between environmental exposures and genetic background rather than from specific genetic variations in hsp70 genes.

Polymorphism in the regulatory sequence of the human hsp70-1 gene does not affect heat shock factor binding or heat shock protein synthesis.

A bi-allelic polymorphism found in the regulatory region of the human heat shock (HS) protein (HSP) hsp70-1 gene, which comprises an A-->C transversion, 3 bp upstream of the HS element (HSE), has been associated with extended HLA haplotypes. In view of the chaperoning and protective functions of Hsp70, we investigated whether this hsp70-1 bi-allelic polymorphism could modulate the stress response, which may relate to enhanced resistance or susceptibility to certain diseases. We compared the basal and HS-induced HS factor (HSF)-binding activity of the two polymorphic HSEs, hsp70-1 mRNA accumulation and HSP expression in two human Epstein Barr virus (EBV)-transformed B cell lines typed for hsp70-1 promoter alleles. Our results suggest that hsp70-1 promoter polymorphism does not influence HSF-binding activity, hsp70 mRNA accumulation or synthesis in human EBV-transformed B cell lines.

HLA class II antigens and T lymphocytes in human nasal epithelial cells. Modulation of the HLA class II gene transcripts by gamma interferon.

BACKGROUND: Nasal polyps are characterized by a proliferation of the epithelial layer of the mucosa, cellular infiltrates and other pathological changes; however the mechanisms involved in polyp pathogenesis remain largely unclear. OBJECTIVES: We have taken two different approaches to study the cellular events involved in nasal polyposis. METHODS: First, through use of immunohistochemical methods, we have studied the expression of HLA class II antigens in epithelial cells of nasal polyps and the distribution of lymphocytes in the epithelium and in the subepithelial layer in patients with clinical conditions, such as asthma, atopy, aspirin intolerance or cystic fibrosis, and in subjects with an absence of concomitant diseases. Second, in order to investigate whether HLA class II expression is controlled at the pre- or post-transcriptional level, we studied the effect of interferon gamma (INF gamma) on epithelial cells in primary culture, which were derived from HLA class II negative and HLA class II positive nasal polyps. Total RNA was extracted from the cells and reverse-transcribed, and the c-DNA corresponding to DR, DP, DQ loci was amplified by PCR. RESULTS: Expression of HLA class II antigens by the epithelia of nasal polyps was more common in the presence rather than in the absence of concomitant asthma, atopy or cystic fibrosis (59% versus 40%). HLA-DR was the only HLA class II antigen expressed in the seven polyps taken from cystic fibrosis patients. The number of CD8+ cells was significantly higher in polyps associated with known clinical conditions and HLA class II antigen expression than it was in 'isolated' polyps and in HLA class II negative polyps. RNA transcripts for at least one or all three HLA-DR, DP and DQ antigens were detected in 10 cultures of the II HLA class II positive polyps. Conversely, 8 of 10 cultures derived from HLA class II negative polyps did not express HLA class II transcripts in the absence of INF gamma. Adding INF gamma (100 U/ml) to the latter cell cultures caused expression of transcripts of one or more HLA class II genes. CONCLUSIONS: We have shown that HLA class II antigens were more frequently detected in polyps of patients with an identified clinical syndrome than in those of asymptomatic subjects. Our results also suggest that IFN gamma regulates expression of HLA class II antigens in airway epithelial cells of the nasal polyps at the transcriptional level, and that cultured cells from nasal polyps represent a suitable model to investigate immune mechanisms involved in diseases such as atopy, asthma and cystic fibrosis.

Evidence of a strong, positive association between atopy and the HLA class II alleles DR4 and DR7.

BACKGROUND: Atopy, with or without associated asthma, provides a useful model for evaluating the genetic factors that control human immune responsiveness. HLA class II gene products are involved in the control of immune responses. OBJECTIVES: We investigated whether susceptibility or resistance to the disease was associated with HLA class II genes. METHODS: Blood samples were obtained from two groups of unrelated European-born white adults: 56 atopic patients (52 of them with asthma) and 39 healthy controls with no personal or familial history of asthma or atopy. Genomic DNA was extracted from peripheral blood lymphocytes. The exons of DQA1, DQB1, DRB and DPB1 genes were selectively amplified by the polymerase chain reaction (PCR) method. Genotyping was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which can recognize allelic variations in the polymorphic exon. RESULTS: We found no significant differences in the frequency of DPB1 alleles between patients and controls. HLA class II DR4 and DR7 alleles were present in 39.2% of the patients and in 2.5% of the healthy subjects (Pc*2 < or = 3.9 10(-3)). Conversely, DQA1*0103 and DQB1*0502 alleles were more frequent in the control subjects. These results confirm a previous study of an extended pedigree, which showed that DR4 and DR7 alleles were absent in all healthy members of the family and were frequently observed in atopic and/or in asthmatic subjects. CONCLUSION: We observed that HLA-DR 4 and DR7 alleles are significantly implicated in their susceptibility to the disease and suggest that this susceptibility is more related to atopy than to specific responses to allergens. According to previous studies, we could also submit that in atopic patients with asthma, DR4 alleles at the least, could be more closely associated with atopy than with asthma per se. Conversely, we suggest that some allelic DQA1 and DQB1 sequences might confer protection against the disease.

A simple and rapid micromethod for genomic DNA extraction from jugal epithelial cells. Application to human lymphocyte antigen typing in one large family of atopic/asthmatic probands.

We describe a rapid and reliable micromethod for DNA isolation from buccal epithelial cells from the interior mouth mucosa. This convenient, noninvasive method could be applied to genetic typing in a small number of cells (about 2000 cells per cheek). We have shown that DNA released by this method is suitable for further amplification by polymerase chain reaction (PCR). Using this protocol, coupled with the PCR-RFLP (restriction fragment length polymorphism) method, we analyzed the allelic sequence diversity of the human lymphocyte antigen (HLA) class II genes in an extended family of 33 persons containing 14 asthmatic or atopic members. Six of eight DQA1 alleles, and 11 DQB1, 20 DPB1, and 10 DR haplotypes could be identified in a single DNA sample. Our results suggest that the DR53 group haplotype is frequently associated with allergic asthma and atopy. The micromethod described here may be useful in genetic epidemiology, especially in family studies involving small children.

Retrieval of precursors for white-type adipose conversion in brown adipose tissue.

A cellular compartment from brown adipose tissue (BAT) of newborn rats was isolated by Percoll-density-gradient centrifugation and was shown to proliferate and to undergo adipose conversion in vitro in primary culture. The features of the effector requirement for adipose conversion as well as the differentiated morphological and biochemical phenotype are almost identical with that of a compartment designated HCF, from white adipose tissue (WAT). A possible role for these precursors from BAT and WAT in the involution of BAT into WAT, on the one hand, and in the development of brown adipose cells among typical WAT deposits, on the other, is discussed.

Characterization of rat preadipocytes from normal rat adipose tissue by their effector response.

We isolated from normal rat adipose tissue, by a Percoll-density-gradient procedure, two populations of adipocyte precursors. These preadipocytes undergo morphological and biochemical adipose conversion in primary culture. For full adipose conversion, these precursor cells, in addition to the adipogenic factor present in fetal-calf serum, require other effectors differentially. One population completes terminal differentiation in the presence of physiological concentrations of insulin. The second population requires a pre-sensitization with isobutylmethylxanthine at a critical period of the culture in order to respond to insulin. The fact that dibutyryl cyclic AMP could not be substituted for isobutylmethylxanthine suggests that the effect of the latter agent is not through its inhibition of particulate phosphodiesterase activity. These two populations further differ in their response to exogenously added haemin. Thus the existence of at least two developmentally regulated rat adipose-precursor compartments is demonstrated.

Characteristics of the rat adipoblast during differentiation in culture.

In the present study, stromal vascular cells were obtained from the inguinal fat pad of 3-day-old rats and layered on a continuous density gradient (Percoll). After centrifugation, it was possible to collect three homogeneous cell populations of different densities and to grow them separately in primary culture. Upon reaching a confluent state two of them particularly (the lighter fractions) converted to adipocytes with a high frequency (90 per cent) in the presence of physiological concentration of insulin (10(-8) M). During the adipose conversion insulin markedly enhanced the activities of lipogenic enzymes. When VLDL (350 micrograms lipids/ml) and heparin (10 IU/ml) were added to the medium, this effect was not potentiated. However VLDL and heparin in presence of insulin increased the triacylglycerol content of the differentiated cells. The heavier fraction did not undergo adipose conversion to the same extent as the lighter ones. It was concluded that the high ability of these precursor cells to convert to adipocytes and their response to low concentration of insulin could be related to the early development stage of the tissue.

[Characterization and differentiation of rat preadipocytes in primary culture]. / Caractérisation et différenciation de préadipocytes de rats en culture primaire.

Using a density gradient medium, we isolated homogeneous cell populations from the inguinal tissue of 3-day old rats. In primary culture we obtained, for the first time, the differentiation of 99% of the cells in the presence of a physiological concentration of insulin (10(-9) M). This model closely mimicked the events occurring during normal mammalian adipose development, i.e. a positive change in beta-adrenergic sensitivity, early induction of lipoprotein lipase, expression of the enzymes involved in the triglyceride systems, and the development of responsiveness to glucagon.

Development under the control of insulin of lipogenic enzymes, lipoprotein lipase, isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture.

In preadipose cellular fractions (I, II and III) isolated by density gradient centrifugation from the inguinal tissue of young rats, we followed the activity of fatty acid synthetase, ATP citrate lyase and lipoprotein lipase during differentiation in culture. 1.5 nM insulin when added at confluence markedly induced the activity of ATP citrate lyase and fatty acid synthetase in the cells derived from the lighter fractions (I and II). The magnitude of this response was 25-50-fold the initial value 15 days after plating. In the cells of the heaviest fraction (III) both enzymes exhibited low activity which was slightly stimulated by the presence of insulin, VLDL and heparin. In contrast, the activity of lipoprotein lipase appeared before confluence in cells from all three fractions and peaked at day 6 after plating. This early emergence was independent of the addition of insulin to the medium. However, insulin slightly enhanced the peak activity in post-confluent cells. The development of cAMP production in response to isoproterenol (100 microM) and to glucagon (0.3 microM) was determined in the cells of fraction II in the same culture conditions. The responsiveness to isoproterenol was present very early in these cells and rose rapidly during the exponential growth phase, reaching a peak value at day 8 after plating. In contrast, the development of glucagon sensitivity occurred only during late differentiation. The stimulatory effect of glucagon was enhanced when VLDL and heparin were added with insulin to the medium.

Differentiation under the control of insulin of rat preadipocytes in primary culture. Isolation of homogeneous cellular fractions by gradient centrifugation.

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10(-9) M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid: CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.

Alteration, by early underfeeding, of cellular multiplication and differentiation in the inguinal fat pads of rats.

The effect of litter size on the incorporation of labeled thymidine (TdR) into DNA was studied in the stromal and the adipocyte fractions of the rat inguinal tissue. In experiment 1 the animal were kept in litters of 18 (UF) or 6 (control) from birth till 10 days. They were injected with [2-14C] TdR at day 3 and killed at 60 minutes, 1, 3 and 7 days post-injection. In experiment 2, the pups were raised in litters of 18 during 3 (RF3), 6 (RF6) or 10 (RF10) days, and distributed again in litters of six. They were injected with [2-14C] TdR or [14CH3]TdR at the beginning of the refeeding and killed as described previously. In all experiments the weight of the inguinal tissue was more reduced than the total body weight. In the UF, the proliferation was markedly reduced in cellular fractions as was the differentiation of stromal cells into adipocytes from six days of underfeeding. In the RF3 and the RF6 there was an attempt to recover the cell number as suggested by the recycling of the degradation products of TdR for DNA synthesis. In the RF10, cell multiplication and differentiation were strongly affected by the length of the deprivation period.

Studies on cell proliferation in inguinal adipose tissue during early development in the rat.

[2-14C] Thymidine was injected into rats aged 3,5 and 10 days, and incorporation of the precursor into deoxyribonucleic acid (DNA) of the inguinal fat tissue was measured for short time periods. Using chromatographic procedures to measure the distribution of thymidine and its metabolites in the soluble fraction of the tissue, degradation of the precursor was found to be similar at all ages. The data indicate that thymidine was more rapidly utilized for DNA synthesis in 3-day-old rats than in older animals. When 14C-thymidine was injected in vivo and adipocytes and stromal cells were then separated from the inguinal tissue of 3-and 5-day-old rats, the incorporation into DNA was significant in both types of cells already 30 min after pulse labeling. Stromal cells took up twice as much of label as the adipocytes. Furthermore, real incorporation into DNA was found in the adipocytes when incubated in vitro in a culture medium supplemented with 14C-thymidine. The possibility is discussed that early in postnatal life adipocytes might synthesize DNA for further cell division.