Entropy-favored human antibody binding reactions with a non-infectious antigen.

Little is known about the thermodynamic properties of human antibodies directed against 'natural' antigen surfaces, possibly due to the complex interactions that are involved. Using an affinity distribution method, we have previously characterized the binding reactions between a major allergen from ragweed, Amb a 1, and serum Amb a 1-specific IgE as a model system. We determined the temperature dependence of these interactions using serum samples from people with established allergic sensitivity to ragweed pollen. Each sample provided evidence for three epitope-specific IgE reactions with extremely high equilibrium binding affinities @ 37 degrees C (10(8) to 10(11)M(-1)). Determining the affinities over a range of temperatures (4-41 degrees C) revealed a favorable exothermic Gibbs free energy change, DeltaG approximately -17.59 (+/- 5.04)kcal/M, comparable to previous reports using monoclonal antibodies produced against well-defined artificial antigens. In contrast to previous studies, in this system there was minimal input from enthalpy: DeltaH approximately -2.41 (+/- 2.32)kcal/M. However, a significant contribution was found from entropic changes: DeltaS approximately 48.98 (+/- 9.20)cal/KM. Human 'secondary antibodies' such as IgE, produced after exposure to 'natural' antigens, are optimized in terms of their high equilibrium binding constants with the antigen (allergen) that induced their production. Thermodynamically this is exemplified by minimal enthalpic (bond formation) concomitant with significant entropic (alignment) contributions to the total free energy change of reaction. These results suggest a high degree of 'complementarity' between the antibody and antigen surfaces in this experimental system, and may be a general guiding principle in the evolution of antibody repertoires by the adaptive immune system.

Search for quantitative trait loci of atopy-associated immune responses using allergen-specific IgG1 as an "endophenotype".

Inherited atopic diseases of humans arise from adverse adaptive humoral responses to noninfectious environmental allergens. We previously reported that allergen-specific IgG1 provides more reliable heritability estimates for responses to allergens than total IgE. Genome scans were done for 91 Caucasian nuclear families with history of atopy for total IgE and IgG1 produced against a common major allergen from house dust mite, Der p 1. Suggestive associations for Der p 1-IgG1 production were found at 7 quantitative trait loci (QTL) (logarithm of the odds, LOD > or = 1.23; p < or = 0.009) with QTL-specific heritabilities of 73%-80%. Scans using total IgE found suggestive associations for 12 QTLs (LOD > or = 1.44; p < or = 0.004), but QTL-specific heritabilities only in the range of 30%-35%. Allergen-specific IgG1 is a suitable "endophenotype" to be used in searches for genes associated with atopy-associated humoral immune responses to common aeroallergens.

Allergen-specific IgG1 provides parsimonious heritability estimates for atopy-associated immune responses to allergens.

Although serum total immunoglobulin E (IgE) is generally elevated in atopic conditions, it is an unreliable trait for dissecting the genetic and environmental components contributing to atopic immune responses, because it can be significantly confounded by demographic factors (age, gender, and race) and clinical status (atopic vs nonatopic). Allergen-specific IgE is a discontinuous trait present only in those with sensitivity to allergens. However, all people will produce allergen-specific immunoglobulin G1 (IgG1), which is elevated among those atopically sensitized to specific allergens. We screened 91 Caucasian nuclear families (N = 367) with medical histories of atopic diseases and used variance components analysis to compare heritability estimates for total IgE and IgG1 produced against the common major allergen from house dust mite Dermatophagoides pteronyssinus (Der p 1). An estimate of total IgE heritability was about 48%, although this was significantly confounded by age, gender, and clinical atopic status. In contrast, Der p 1-IgG1 demonstrated a significant inherited component of about 62% that was not influenced by age, gender, or clinical status. For genetic studies of atopic humoral responses, allergen-specific IgG1 may be a more reliable quantitative trait than serum IgE. Moreover, atopy is an inherited deregulation of immune responses to noninfectious antigens, involving antibody isotypes other than IgE.

Random allergen-specific IgE expression in atopic families: evidence for inherited "stochastic bias" in adverse immune response development to non-infectious antigens.

The complex inherited human atopic diseases are associated with adverse IgE-mediated immune responses, notably allergen-specific IgE that presumably involves the input from one or more genes. However, gene searches have met with limited success, possibly because a causally direct gene input-trait outcome assumption is not valid for these immune responses. To test this assumption, we determined the probability distributions of quantitative IgE responses associated with atopy, and used these to determine the statistical interdependence among first-degree relatives (parent-child and sibling-sibling) from families with history of atopic asthma (total available N=1099). Each person was screened for asthma history, pulmonary responses by spirometry and atopic immune responses using serum total IgE and skin prick tests (SPT) to 14 allergens. Heritability estimates were made by variance components analysis for quantitative IgE traits. The serum total IgE distribution comprised statistically independent sub-sets when individuals were categorized as either SPT [-] or SPT [+], reflecting contributions from non-pathology associated basal IgE and pathology-associated allergen-specific IgE. However, heritability estimates were significant only for basal IgE, while total allergen-specific IgE production was a random variable independent of inheritance. Genes for specific IgE-mediated responses are not obligately inherited. Rather, gene products that modulate underlying stimulus-response coupling interactions and alter the probabilities influencing adverse immune responses are inherited, but an individual's specific pathologic outcome is a random variable. These results support a model of "stochastic bias" that "skews" an immune response to non-infectious antigens among people with an inherited predisposition for atopy.

CD14 promoter polymorphisms in atopic families: implications for modulated allergen-specific immunoglobulin E and G1 responses.

BACKGROUND: CD14 promoter DNA sequence polymorphisms for the endotoxin receptor gene have been implicated in modulating allergen-specific immunoglobulin (Ig)E responses in randomly selected individuals with atopy. We sought to determine if a single nucleotide polymorphism in the CD14 promoter region is associated with atopy in atopic families, and to assess its influence on serum levels of CD14 and allergen-specific IgE and IgG1 responses. METHODS: We screened 367 members of 91 Caucasian nuclear families with a history of asthma for pulmonary function by spirometry, including methacholine challenge to detect bronchial hyperreactivity, and atopy by serum total IgE and skin prick test to 14 allergens. The CD14 promoter single nucleotide polymorphism was analyzed in DNA isolated from peripheral blood mononuclear cells to identify C/C, C/T and T/T genotypes. Serum tests were done for soluble CD14 (sCD14) and dust mite-specific antibody (Der p 1-IgG1). RESULTS: Serum sCD14 levels were not associated with clinical phenotypes (asthma, bronchial hyperreactivity or atopy). However, sCD14 levels were inversely related to both allergen-specific IgE and Der p 1-IgG1 production, but only among those with evidence of atopic sensitization. Linear regression analysis, accounting for random family effects, demonstrated a higher production of allergen-specific IgE or Der p 1-IgG1 associated with the T/T genotype and a lower level of specific IgE and IgG1 production associated with sCD14 levels. CONCLUSIONS: An element of the innate immune system (CD14) has profound effects upon modulating the acquired allergen-specific immunoglobulin responses among those with an inherited atopic predisposition.

Evidence that negative feedback between antibody concentration and affinity regulates humoral response consolidation to a non-infectious antigen in infants.

The dynamics of human antigen-specific immunoglobulin (Ig) responses in early life are not well characterized. We have previously observed an inverse relationship between allergen-specific Ig concentration and allergen-Ig-binding affinity in allergen-sensitive atopic adults, suggesting a possible feedback relationship between these variables. We prospectively studied children (6 months to 6 years) with and without atopic sensitization to the Der p 1 major allergen. Experimental results showed the following trends. (1) In both study groups, there was little change with age in average Der p 1-specific Ig (IgG1 or IgE) concentrations or allergen-Ig-binding affinities, and concentrations and affinities were independent. (2) Among individuals, however, there was a negative correlation between Ig concentration changes and affinity changes with age. (3) The rate of increase with age of the non-atopic Der p 1-IgG1 total binding capacity (Ig concentration x Ig affinity) paralleled that for the atopic Der p 1-IgE total binding capacity, and there was a comparable 'consolidation' of responses with age reflected by a narrowing of the variance of total binding capacity values. Except for the Ig classes involved, development of a humoral response to a non-infectious allergen is similarly regulated in atopic and non-atopic children, with Ig total binding capacity as the key regulatory variable. These results also suggest that there is a time-dependent feedback relationship between Ig concentrations and affinities that establishes an optimal Ig total binding capacity for a given environmental 'antigen load'. A theoretical model is proposed to account for this relationship.

Evidence for two independent distributions of serum immunoglobulin E in atopic families: cognate and non-cognate IgE.

Genetic studies of IgE-mediated atopic disease have produced conflicting results, due largely to variable phenotype definitions. Total IgE concentrations and 14 allergen skin prick tests (SPT) were determined in 1099 members of families with history of atopy. Log10 [Total IgE] values were normally distributed in both atopic (SPT [+]) and non-atopic (SPT [-]) groups. The mean Log10 [Total IgE] value was higher in the atopic group, although the standard deviations of the distributions were the same. The mean Log10 [Total IgE] value of the non-atopic distribution was subtracted from the individual Log10 [Total IgE] values of the atopic group giving an allergen-specific fraction. There was a strong positive correlation between the specific IgE fraction and the number (#) SPT [+] results, defined as Cognate IgE. Among the atopics, subtracting the Cognate IgE value from total IgE yielded Non-Cognate IgE. The Cognate and Non-Cognate IgE distributions were statistically uncorrelated. Evidence is presented for two serum IgE fractions that are statistically and physiologically independent of one another in atopic families; a Cognate IgE fraction associated with atopic sensitization and a Non-Cognate IgE fraction unrelated to atopic disease. Elevated serum IgE is a consequence, not a predisposing cause, of allergen sensitization.

Robustness into advanced age of atopy-specific mechanisms in atopy-prone families.

We evaluated atopy-associated parameters in 1,099 people (aged 6-84 years) from families with history for atopy. All were tested for serum total immunoglobulin E (IgE) and allergen sensitivity by skin prick test. Specific IgE tests were done in randomly selected families. There was a decline with age in serum total IgE values, and relative atopy "incidence rates" were slightly lower among those older than 60 years. However, there was no change with age in sensitivity or severity of atopy. Among those sensitized to ragweed (Ambrosia artemisilfolia), there was no age-associated change in IgE levels specific to Amb a 1, a major allergen extracted from ragweed, and no change in the binding affinity of IgE for the Amb a 1 allergen. Among families with atopic histories, the underlying atopic mechanisms are particularly robust, and the atopic propensity remains into advanced age. In addition, established atopic responses may be focused in an immune system compartment either independent of or minimally influenced by T-cell activity.

Allergen skin test reaction patterns in children (

BACKGROUND: Genetic studies of atopy rely upon evidence of abnormal IgE production, usually elevated total IgE or skin prick test (SPT) reactions. However, these measures may change with subject age. METHODS: We screened 1,099 members of atopic families (aged 6-87 years) by serum total IgE and SPT for 14 allergens. For those SPT negative, we screened for Amb a 1- and Der p 1-specific IgE. Der p 1 IgE-Der p 1 allergen binding affinities were done on randomly selected subjects. RESULTS: There were significantly fewer atopics 10 years old (75.8%) based upon any SPT-positive result. Children 10 years old = 82.3%). Among those SPT-positive for house dust mite extract, there was a positive correlation between Der p 1 binding affinity and the wheal area of the house dust mite extract. There was a positive correlation between the number of SPT-positive reactions and total IgE for both age groups. However, there was only a significant relationship between SPT-positive wheal area and total IgE for those >10 years old and no apparent relationship between wheal area and total IgE for those

Variable binding affinities for allergen suggest a 'selective competition' among immunoglobulins in atopic and non-atopic humans.

Atopy is a persistent, aberrant humoral response to certain classes of proteins (allergens) characterized by the presence of allergen-specific IgE. Yet, in both atopic and non-atopic individuals, allergen-specific responses involving the IgA and IgG subclasses have been observed, which evidence does not support models suggesting inherited differences in sensitivity to certain protein classes. Using the major ragweed component Amb a 1 as a model allergen, we assessed the humoral responses in three groups of unrelated donors: (A) atopic, ragweed sensitive; (B) atopic, but not ragweed sensitive; (C) non-atopic. As expected, Amb a 1-specific IgE was present in group A only. However, there were essentially no differences in the relative proportions of Amb a 1-specific IgA(1,2) and IgG(1-4) among the groups. We also determined the Amb a 1 binding affinities for IgG(1) and IgG(4) in the three groups, and compared these to Amb a 1-specific IgE binding affinities in group A. Group A donors' Amb a 1-IgE had extremely high affinities (10(8) to 10(11)M(-1)), but their Amb a 1-IgG(1) and Amb a 1-IgG(4) affinities were significantly lower (10(7) to 10(10)M(-1)). The average IgG(4) binding affinities in groups B and C were slightly higher than that of IgG(4) in group A, although not statistically significant. However, the IgG(1) affinity for Amb a 1 among group C, non-atopic donors was significantly elevated and comparable to the IgE affinity observed in group A, ragweed atopics. Inhibition studies with allergen-specific IgE-free serum showed that all isotypes recognized the major epitopes seen by IgE. These results suggest that there may be a "selective competition" among isotypes for allergens that is driven by the ability to produce high affinity, allergen-specific immunoglobulins.