Analysis of the expression of immunoglobulins throughout lactation suggests two periods of immune transfer in the tammar wallaby (Macropus eugenii)
Introduction
Marsupial young are born in an altricial state of development. Only the organ systems regarded as essential for birth and perinatal survival are well developed at parturition. The immune system, however, is under-developed and naïve at birth. Pouch young do not develop adult-like immune systems until just prior to first leaving the pouch, at a period called the switch phase (Baker et al., 2005, Basden et al., 1997, Belov et al., 2002b, Block, 1960, Deane and Cooper, 1988, Old and Deane, 2000). In the tammar wallaby (Macropus eugenii) this is at 90 days of age (Basden et al., 1997) and in the brushtail possum (Trichosurus vulpecula), 70 days (Belov et al., 2002b). In addition, the pouch itself contains potentially pathogenic organisms at all stages of the reproductive cycle (Deakin and Cooper, 2004, Old and Deane, 1998). The secretion of antibodies and other immune factors in the milk of the mother (Adamski and Demmer, 1999, Adamski and Demmer, 2000, Adamski et al., 2000, Deane and Cooper, 1988, Demmer et al., 1999), confers protection to these immunologically naïve young during the early post-partum period.
The marsupial lactation cycle differs from those seen in eutherian species. Lactation is divided into four phases. Phase 1 covers gestation and lasts for approximately 26.5 days in the tammar (Trott et al., 2003, Tyndale-Biscoe and Renfree, 1987). During this phase, all four glands will develop and produce milk (Hendry et al., 1998, Trott et al., 2003). Phase 2A begins with the birth of the young. The tammar neonate makes it way from the cloaca to the pouch, where it will attach to one of the four teats. The remaining teats will then regress. During phase 2A, the pouch young is permanently attached to this teat. Phase 2B or the switch phase begins at 120 days in the tammar (Hendry et al., 1998, Trott et al., 2003, Tyndale-Biscoe and Renfree, 1987). The tammar neonate is still within the pouch, but begins to suckle intermittently. Milk produced during phases 2A and 2B is low in fat and protein, but high in carbohydrates (Hendry et al., 1998, Trott et al., 2003). Phase 3 begins at 180 days and the tammar neonate begins to leave the pouch (Trott et al., 2003, Tyndale-Biscoe and Renfree, 1987). The young begins to eat herbage and it will still suckle, albeit less frequently and larger volumes. Milk produced during this last stage is higher in volume, protein and fat but lower in carbohydrates than milk secreted in the earlier phases (Hendry et al., 1998, Trott et al., 2003). This phase concludes with weaning and involution at 300–350 days in the tammar (Trott et al., 2003, Tyndale-Biscoe and Renfree, 1987).
The immunological significance of milk is a well-studied area in eutherians. Immunoglobulins are usually secreted in high amounts in the colostral milk of eutherians. These immunoglobulins may be taken up by the eutherian neonate's gastrointestinal system and are absorbed into their circulation in some species, thus providing passive immunity. This absorption is limited by the time taken for the gastrointestinal system's epithelium to close, and ranges from 24 h in some ungulates, to up to 19 days in rodents (Kolb, 2002). However, in marsupials absorption of immunoglobulins, other proteins and possibly cells continues throughout the entire lactation period (Cockson and McNeice, 1980, Green and Renfree, 1982, Yadav, 1971, Young et al., 1997). Metatherian immunoglobulin levels in milk are much lower than those seen in eutherians (Basden et al., 1997, Deane et al., 1990). Consequently, marsupials must rely upon more efficient uptake and different immunoglobulin isotypes to help meet the immunological challenges of the young (Deane et al., 1990). The brushtail possum has two periods of immune transfer during lactation and these periods coincide with the periods of greatest immune challenge for the pouch young—i.e. birth and leaving the pouch (Adamski and Demmer, 1999, Adamski and Demmer, 2000, Adamski et al., 2000, Demmer et al., 1999). These two phases of immune transfer in the mammary gland of the brushtail possum has been demonstrated for not only immunoglobulins (Adamski and Demmer, 1999, Adamski and Demmer, 2000) but also other immune components such as the neonatal Fc alpha receptor (FcRn) (Adamski et al., 2000) and the iron regulatory proteins ferritin and transferrin (Demmer et al., 1999). This pattern of differential expression occurs in the gastrointestinal tract of the brushtail possum young late in lactation (Western et al., 2003). Consequently, differential regulation of expression of immune components in both the mammary gland of the mother and the gastrointestinal tract of the young may help the marsupial neonate to survive periods of immune stress while still relatively immunocompromised.
In this study, we have identified and examined the expression of immunoglobulin heavy and light chains, the J chain, the polymeric immunoglobulin receptor (pIgR), the neonatal Fc receptor (FcRn) and the IgA secreting cell specific chemokine CCL28 in the mammary gland of the tammar wallaby. This work shows that the tammar wallaby possesses all heavy and light chain isotypes previously identified in marsupials (IgA, IgE, IgG, IgM, Igκ and Igλ). In addition, we have examined the functional regions of these immunoglobulins and their receptors. While there was a high level of conservation present in many functional motifs of immunoglobulins and their receptors, there were some non-conservative substitutions in a motif important in pIgR binding in IgA that may affect polymerisation and transcytosis of this mucosal immunoglobulin (Aveskogh and Hellman, 1998, Aveskogh et al., 1999, Belov et al., 1998, Belov et al., 1999a, Belov et al., 1999b, Belov et al., 1999c, Belov et al., 2001, Belov et al., 2002a, Lucero et al., 1998, Miller et al., 1999), and in an FcRn-binding affinity motifs in IgG. Two distinct periods of increased immune expression were observed for most of these immune components in the tammar mammary gland. This suggests that differential regulation of immunoglobulins and their receptors is a generic marsupial mechanism and may be an important defence strategy for the survival of pouch young.
Section snippets
Animals
Tissue was obtained from a colony of tammar wallabies (M. eugenii) kept in open grassy yards at the Victorian Institute of Animal Science, Attword, Victoria, Australia. Food and water was provided ad libertum. All procedures were carried out according the guidelines of the experimentation ethics of the Victorian Institute of Animal Science.
Expressed sequence tags (EST) library construction
Twenty cDNA libraries were prepared using tammar wallaby mammary gland RNA from day 23 of pregnancy (n = 4), from lactating glands at day 130 (n = 4), day 260 (n =
Identification and characterization of sequences
From the tammar EST library, partial sequences of Cμ, Cα, Cγ, Cɛ, FcRn, J chain, pIgR and full length sequences of Igκ and Igλ were isolated. Isolated tammar Cμ, Cα, Cγ and Cɛ sequences were compared with known marsupial sequences from the brushtail possum and the grey short-tailed opossum. The tammar sequence for Cγ (EF599608) was deduced from a single clone and was 740 nucleotides in length, which translated to an open reading frame (ORF) of 246 residues in length. When this was compared to
Discussion
Milk has roles in not only nutrition, but also immune defences that are crucial for neonatal survival. In this study we have demonstrated differential regulation of three of these immunoglobulin heavy chain isotypes, corresponding light chains, receptors (pIgR, FcRn and J chain) and an associated chemokine (CCL28) in a macropod marsupial, the tammar wallaby, across the lactation cycle. These results suggest that differential regulation of immunoglobulins throughout the lactation cycle in
Conclusion
In conclusion, in this study we have isolated and characterized the heavy chain constant regions of immunoglobulin isotypes, their κ and λ light chains, the receptor FcRn, J chains and the chemokine CCL28 in the tammar wallaby. While there were high levels of conservation of functional areas of these molecules in the tammar and other marsupials, we did identify some motifs, which were poorly conserved and may have potential effects on expression or function of these immunoglobulins. The tammar
Acknowledgements
This work was supported by the CRC for Innovative Dairy Products, Australia and an Australian Postgraduate Award from the University of Sydney.
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