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. 2009 Aug 11;5(6):784–787. doi: 10.1098/rsbl.2009.0435

Mhc polymorphisms fail to explain the heritability of phytohaemagglutinin-induced skin swelling in a wild passerine

Camille Bonneaud 1,2,*, Janet S Sinsheimer 3, Murielle Richard 1, Olivier Chastel 2, Gabriele Sorci 1,†
PMCID: PMC2827992  PMID: 19671600

Abstract

Genetic estimates of the variability of immune responses are rarely examined in natural populations because of confounding environmental effects. As a result, and because of the difficulty of pinpointing the genetic determinants of immunity, no study has to our knowledge examined the contribution of specific genes to the heritability of an immune response in wild populations. We cross-fostered nestling house sparrows to disrupt the association between genetic and environmental effects and determine the heritability of the response to a classic immunological test, the phytohaemagglutinin (PHA)-induced skin swelling. We detected significant heritability estimates of the response to PHA, of body mass and tarsus length when nestlings were 5 and 10 days old. Variation at Mhc genes, however, did not explain a significant portion of the genetic variation of nestling swelling to PHA. Our results suggest that while PHA-induced swelling is influenced by the nest of origin, the importance of additive genetic variation relative to non-additive genetic variation and the genetic factors that influence the former in wild populations still need to be identified for this trait.

Keywords: major histocompatibility complex, phytohaemagglutinin-induced skin swelling, cross-fostering, heritability, house sparrow

1. Introduction

Genetic contributions to the phenotypic variability of parasite resistance and immune response have mainly been demonstrated in domestic or laboratory species (Lillehoj et al. 2007). The genetic variation underlying those traits is however harder to detect in natural populations (Kilpimaa et al. 2005), in part because of environmental variance (Sorci et al. 1997), but also because fitness traits have lower heritabilities than non-fitness traits (Mousseau & Roff 1987).

The phytohaemagglutinin (PHA)-induced skin swelling test is classically used in vivo in immunoecological studies and provokes the infiltration and/or proliferation of several types of immune cells (Martin et al. 2006). When added to cultures of T lymphocytes, PHA can bind T-cell receptors and initiate a complex signalling cascade (Kuno et al. 1986; Chilson & Kellychilson 1989), resulting in their proliferation and differentiation independently of their specificity or clonal origin (Licastro et al. 1993). Importantly, chicken lines have been shown to differ in swelling intensity in reaction to PHA, demonstrating significant genetic variation for this response (Sundaresan et al. 2005).

Since major histocompatibility complex (MHC) molecules are not only important in foreign antigen detection but also serve key roles in the activation and proliferation of T lymphocytes (Gur et al. 1999), several studies have investigated their role in the response to PHA (for e.g. Rubini et al. 1992; Kimball et al. 2002). In fact, Mhc genes have previously been found to be associated with the phenotypic variability of this response in chickens and in a wild passerine (Taylor et al. 1987; Bonneaud et al. 2005). Here, we decomposed the phenotypic variability of the response to PHA in its genetic and environmental components by cross-fostering nestling house sparrows (Passer domesticus) between synchronous broods. We then investigated the sources of variation of this response and provide, to our knowledge, the first examination of the implication of Mhc class I genes in the heritability estimates of this trait in a natural population.

2. Material and methods

(a). Field and laboratory procedures

This study was conducted during spring 2003 at the Center d'Etude Biologique de Chizé (France) on a nest-box population of house sparrows. We analysed phenotypic data for 182 chicks across 61 broods; genotypic and phenotypic data were available for 150 chicks across 55 broods. For a total of 42 broods, two chicks were randomly cross-fostered between synchronous broods at two days of age (n = 84 chicks, 56 surviving to measuring). To maximize statistical power, all 61 broods were used in analyses; results were qualitatively similar when only the 42 experimental broods were included. Body mass (± 0.1 g) and tarsus length (± 0.01 mm) were measured on 5 and 10 day old chicks. PHA was intradermally injected at day 10 (0.025 mg in 0.04 ml phosphate buffered saline; Sigma L8754) and measured after 24 h as described in Bonneaud et al. (2005). Blood was sampled from the brachial vein of 10 days old chicks (approx. 50 µl) and of their parents (approx. 150 µl), stored in buffer/EDTA 2 mM and kept frozen at −20°C for subsequent DNA extraction.

We evaluated the allelic diversity of all chicks at the most variable Mhc class I gene family using the PCR-based denaturing gradient gel electrophoresis method (Bonneaud et al. 2004). Adults and chicks were genotyped using seven microsatellite markers as described in Bonneaud et al. (2006). We compared the microsatellite genotypes of both parents and chicks to verify paternity and identify the chicks sired from extra-pair mating. Since the microsatellite loci were highly polymorphic, we set the detection probability of extra-pair young at 0.99. Extra-pair young were accounted for and maintained in all analyses.

(b). Statistical analyses

Variance components for each trait were estimated in Mendel (Lange et al. 2001) using the Polygenic and quantitative trait loci option, which was also designed for classical heritability studies. This model treats the data as multivariate normal. In principle, the phenotypic variance could be partitioned into independent environmental (VE), shared nest of rearing environmental/household (VH), additive genetic (VA), dominance genetic (VD) and maternal/early environmental components (VM) (see the electronic supplementary material, methods). The design of this study precludes separating VA, VD and VM, so that only VA is included in the model. Heritability was calculated as h2 = VA/VP, where VP is the observed phenotypic variance and the sum of all the components.

The statistical significance of heritability estimates were determined by calculating the difference of the max log-likelihoods obtained from the models with and without the additive effect (see the electronic supplementary material, methods; Self & Liang 1987). The statistical significance of the rearing environment (VH) was estimated in the same way. We investigated the contribution of: (i) the total number of Mhc alleles in chicks, and (ii) the presence or absence of the seven most common Mhc alleles in the population (Bonneaud et al. 2006), to the variability of the response to PHA using a measured genotype approach that treats the genotype as a fixed effect in the mean part of the variance component model (Boerwinkle et al. 1986; Lange et al. 2005) and by incorporating each factor separately into the model. Statistical significance was determined using a standard Z-test.

3. Results

Body mass, tarsus length and response to PHA all displayed significant nest of origin effects (p < 0.001 for measures of body mass and tarsus length and p = 0.029 for the response to PHA; table 1), suggesting that they were significantly heritable. As expected, nest of rearing effects increased with chick age for both body mass and tarsus length. There was a significant effect of nest of rearing for body mass and tarsus length only (p < 0.001), not for the response to PHA (p = 0.21). As a result, both shared origin and the rearing environment affected chick development and condition, although PHA-induced swelling was mostly dominated by nest of origin effects.

Table 1.

Variance components for nestling body mass, tarsus length and PHA-induced skin swelling. (VA is conflated by non-additive effects (dominance and maternal/early rearing) so that h2 is more a reflection of broad sense than narrow sense heritability. VE is the variance components due to environmental effects and error variance and VH, the variance components due to a nest of rearing effect.)

VA
 VH
 VE
 h2
trait estimate s.e. estimate s.e. estimate s.e. estimate s.e.
mass at day 5 5.51 1.51 0.10 0.16 0.80 1.00 0.86 0.13
mass at day 10 5.35 2.03 3.21 1.04 0.88 1.26 0.57 0.19
tarsus length at day 5 1.32 0.38 0.03 0.05 0.28 0.25 0.81 0.22
tarsus length at day 10 0.98 0.27 0.48 0.18 0.09 0.16 0.63 0.11
PHA-induced skin swelling 1771.5 1088.3 344.0 467.5 1712.3 711.7 0.46 0.19
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Mhc genes, however, assessed either as the total number of alleles, or the presence or absence of the seven most common alleles, did not explain a significance portion of the phenotypic variability of the response to PHA (table 2).

Table 2.

Contribution of the total number of Mhc alleles detected in house sparrow chicks, as well as the presence of the seven most common alleles (absence of the allele as the reference state), to the estimates of their swelling response to PHA. (We indicate the standard error (s.e.) of the estimate of the effect of each Mhc variable on the response to PHA, the coefficient of variation (CV), the heritability of the response to PHA calculated for each model, as well as the per cent heritability explained by each Mhc variable. We also indicate the population frequency of each Mhc allele. p-values were calculated for a standard normal probability curve.)

allele frequency estimate s.e. CV p-value residual h2 (s.e.) % heritability explained
number of Mhc alleles −2.22 2.84 1.28 0.22 0.46 (0.15) 0.43
Mhc allele a158 0.50 −8.04 10.44 1.40 0.22 0.42 (0.16) 9.72
Mhc allele a172 0.28 −14.89 10.54 0.71 0.08 0.43 (0.16) 6.26
Mhc allele a161 0.23 −7.47 12.46 1.67 0.27 0.46 (0.15) 1.30
Mhc allele a151 0.22 0.88 12.49 14.27 0.47 0.44 (0.17) 5.18
Mhc allele a165 0.21 11.33 12.32 1.09 0.18 0.48 (0.15) 0.00
Mhc allele a163 0.20 −16.22 12.70 0.78 0.10 0.41 (0.16) 12.3
Mhc allele a123 0.16 17.09 10.67 0.63 0.06 0.39 (0.16) 16.5
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4. Discussion

Nestling skin swelling displayed a significant nest of origin effect and was the only nestling parameter not to be affected by the rearing environment. Significant genetic variation in the response to PHA has rarely been demonstrated in wild bird populations (Cichón et al. 2006). Since heritability estimates of immunocompetence have, however, been shown to exhibit lower values in poor-quality compared with high-quality environments (De Neve et al. 2004), it is possible that rearing conditions were, in our study, sufficiently homogeneous between nests to allow for the expression of a nest of origin effect on this trait.

We found that the heritability of nestling response to PHA was not attributable to variation in Mhc genes. One possibility is that the lack of association results from the timing of measure of the response. Indeed, Taylor et al. (1987) found that Mhc genotypes were significantly associated with PHA-induced wattle swelling in chicken, but only in the later phases of the response (≥72 h post-injection). This delay may reflect the initiation of T-cell proliferation (Martin et al. 2006).

Another explanation arises from the inclusion of non-additive genetic effects (dominance and maternal/early rearing effects) in the nest of origin effect that we detected. Recently, Pitala et al. (2007) used an animal model to distinguish additive from non-additive genetic effects on the response to PHA in collared flycatchers (Ficedula albicollis) and failed to show any evidence of a narrow sense heritable response to PHA. If PHA-induced swelling is mostly dominated by maternal effects, it is not surprising that our heritability estimate of nestling response to PHA, which includes early maternal influences, could not be explained by genetic variation.

We previously showed an association between the Mhc allele a172 and response to PHA in this species (Bonneaud et al. 2005). However, because this association is unlikely to reflect a direct involvement of MHC molecules in the physiological processes elicited by PHA (Licastro et al. 1993; Bonneaud et al. 2005), the pattern we detected here could also result if other genes have stronger effects on this trait. In accordance with this hypothesis, responsiveness to PHA has been found to involve genes that are not tightly linked to the Mhc (Gasser et al. 1978; Morrow & Abplanalp 1981).

In any case, we cannot eliminate the possibility that, given our sample size, we failed to detect small effects. In fact, we only have 80 per cent power to detect an allelic effect that explains 20 per cent or more of the genetic variance (Purcell et al. 2003). Our study underscores the need for a precise examination of the role of genetic and environmental factors in the transmission of resistance and immunocompetence between generations, in order to gain a better understanding of how individuals optimize the fitness of their offspring.

Acknowledgements

All procedures conducted in this study adhered to guidelines stipulated by laws of animal research in the country in which the research was undertaken and to those outlined by ASAB in their ethics statement on the use of animals for research and teaching. No animals suffered any obvious consequences as a result of being used in this study.

We thank Miguel Alcaide and Andy Russell for useful comments and Céline Chastel for help in the field. Financial support came from the Ministère de la Recherche (bourse doctorale to C.B. and ACI Jeunes Chercheurs to G.S.; France), from l'Agence Nationale de la Recherche (ANR-05-BLAN-0265-01; France) and from the United States Public Health Service grants MH59490 and GM53275.

References

  1. Boerwinkle E., Chakraborty R., Sing C. F.1986The use of measured genotype information in the analysis of quantitative phenotypes in man. I. Models and analytical methods. Ann. Hum. Genet. 50, 181–194 (doi:10.1111/j.1469-1809.1986.tb01037.x) [DOI] [PubMed] [Google Scholar]
  2. Bonneaud C., Sorci G., Morin V., Westerdahl H., Zoorob R., Wittzell H.2004Diversity of Mhc class I and IIB genes in house sparrows (Passer domesticus). Immunogenetics 55, 855–865 (doi:10.1007/s00251-004-0648-3) [DOI] [PubMed] [Google Scholar]
  3. Bonneaud C., Richard M., Faivre B., Westerdahl H., Sorci G.2005An Mhc class I allele associated to the expression of T-dependent immune response in the house sparrow. Immunogenetics 57, 782–789 (doi:10.1007/s00251-005-0046-5) [DOI] [PubMed] [Google Scholar]
  4. Bonneaud C., Perez-Tris J., Federici P., Chastel O., Sorci G.2006Major histocompatibility alleles associated with local resistance to malaria in a passerine. Evolution 60, 383–389 [PubMed] [Google Scholar]
  5. Chilson O. P., Kellychilson A. E.1989Mitogenic lectins bind to the antigen receptor on human-lymphocytes. Eur. J. Immunol. 19, 389–396 (doi:10.1002/eji.1830190225) [DOI] [PubMed] [Google Scholar]
  6. Cichón M., Sendecka J., Gustafsson L.2006Genetic and environmental variation in immune response of collared flycatcher nestlings. J. Evol. Biol. 19, 1701–1706 (doi:10.1111/j.1420-9101.2006.01110.x) [DOI] [PubMed] [Google Scholar]
  7. De Neve L., Soler J. J., Perez-Contreras T., Soler M.2004Genetic, environmental and maternal effects on magpie nestling-fitness traits under different nutritional conditions: a new experimental approach. Evol. Ecol. Res. 6, 415–431 [Google Scholar]
  8. Gasser D. L., Winters B. A., Balabanmalenbaum G.1978Evidence for a gene in rats affecting lymphocyte responsiveness to PHA. Immunogenetics 7, 149–154 (doi:10.1007/BF01844000) [DOI] [PubMed] [Google Scholar]
  9. Gur H., Geppert T. D., Wacholtz M. C., Lipsky P. E.1999The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction. Cell. Immunol. 191, 105–116 (doi:10.1006/cimm.1998.1417) [DOI] [PubMed] [Google Scholar]
  10. Kilpimaa J., Van de Casteele T., Jokinen I., Mappes J., Alatalo R. V.2005Genetic and environmental variation in antibody and T-cell mediated responses in the great tit. Evolution 59, 2483–2489 [PubMed] [Google Scholar]
  11. Kimball P., Verbeke S., Shiffman M.2002Proliferative suppression and class I insufficiency on peripheral blood cells from hepatitis C patients overcome by exogenous cytokines or high-dose mitogen. Clin. Immunol. 103, 317–323 (doi:10.1006/clim.2002.5217) [DOI] [PubMed] [Google Scholar]
  12. Kuno M., Goronzy J., Weyand C. M., Gardner P.1986Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper Tlymphocytes. Nature 323, 269–273 (doi:10.1038/323269a0) [DOI] [PubMed] [Google Scholar]
  13. Lange K., Cantor R., Horvath S., Perola M., Sabatti C., Sinsheimer J., Sobel E.2001Mendel version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets. Am. J. Hum. Genet. 69, 1886 [Google Scholar]
  14. Lange K., Sinsheimer J. S., Sobel E.2005Association testing with Mendel. Genet. Epidemiol. 29, 36–50 (doi:10.1002/gepi.20073) [DOI] [PubMed] [Google Scholar]
  15. Licastro F., Davis L. J., Morini M. C.1993Lectins and superantigens membrane interactions of these compounds with T-lymphocytes affect immune responses. Int. J. Biochem. 25, 845–852 (doi:10.1016/0020-711X(93)90239-B) [DOI] [PubMed] [Google Scholar]
  16. Lillehoj H. S., Kim C. H., Keeler C. L., Zhangt S.2007Immunogenomic approaches to study host immunity to enteric pathogens. Poult. Sci. 86, 1491–1500 [DOI] [PubMed] [Google Scholar]
  17. Martin L. B., Han P., Lewittes J., Kuhlman J. R., Klasing K. C., Wikelski M.2006Phytohemagglutinin-induced skin swelling in birds: histological support for a classic immunoecological technique. Funct. Ecol. 20, 290–299 (doi:10.1111/j.1365-2435.2006.01094.x) [Google Scholar]
  18. Morrow P. R., Abplanalp H.1981Genetic control of lymphocyte-T mitogenesis in chickens. Immunogenetics 13, 189–200 (doi:10.1007/BF00350785) [DOI] [PubMed] [Google Scholar]
  19. Mousseau T. A., Roff D. A.1987Natural-selection and the heritability of fitness components. Heredity 59, 181–197 (doi:10.1038/hdy.1987.113) [DOI] [PubMed] [Google Scholar]
  20. Pitala N., Gustafsson L., Sendecka J., Brommer J. E.2007Nestling immune response to phytohaemagglutinin is not heritable in collared flycatchers. Biol. Lett. 3, 418–421 (doi:10.1098/rsbl.2007.0135) [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Purcell S., Cherny S. S., Sham P. C.2003Genetic power calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 19, 149–150 (doi:10.1093/bioinformatics/19.1.149) [DOI] [PubMed] [Google Scholar]
  22. Rubini M., Selvatici R., Orlando P., Balugani S., Balboni A., Gandini E.1992Altered proliferative kinetics in PHA-activated human lymphocytes-T treated with the anti-HLA class-I monoclonal-antibody 01.65. Cell Prolif. 25, 405–414 (doi:10.1111/j.1365-2184.1992.tb01450.x) [DOI] [PubMed] [Google Scholar]
  23. Self S. G., Liang K. Y.1987Asymptotic properties of maximum likelihood estimators and likelihood ratio tests under non-standard conditions. J. Am. Stat. Assoc. 82, 605–610 (doi:10.2307/2289471) [Google Scholar]
  24. Sorci G., Moller A. P., Boulinier T.1997Genetics of host-parasite interactions. Trends Ecol. Evol 12, 196–200 (doi:10.1016/S0169-5347(97)01056-2) [DOI] [PubMed] [Google Scholar]
  25. Sundaresan N. R., et al. 2005Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response. Vet. Immunol. Immunopathol. 108, 373–385 (doi:10.1016/j.vetimm.2005.06.011) [DOI] [PubMed] [Google Scholar]
  26. Taylor R. L., Cotter P. F., Wing T. L., Briles W. E.1987Major histocompatibility (b) complex and sex effects on the phytohemagglutinin wattle response. Anim. Genet. 18, 343–350 [DOI] [PubMed] [Google Scholar]

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