The eosinophil is a leukocyte whose intracellular mediators are considered to play a
central role in the pathogenesis of allergic diseases, including allergic asthma, allergic
rhinitis and atopic dermatitis, and which is also involved in immunological responses to
parasites. Eosinophil differentiation and maturation from bone marrow progenitors is
regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T
lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent
chemoattractant for circulating and tissue eosinophils, and the production of this
chemokine promotes eosinophil infiltration and accumulation within sites of allergic
inflammation.¶
Eosinophils obtained from inflammatory tissues and secretions display an altered
phenotype in comparison to peripheral blood eosinophils, with increased surface
expression of major histocompatibility complex (MHC) proteins and adhesion
molecules (Hansel et al., 1991), and migration across the microvascular endothelium
may also increase their capacity to generate an oxidative burst (Walker et al., 1993;
Yamamoto et al., 2000). Eosinophils are phagocytic cells, and have been shown to
present simple (no requirement for intracellular processing) and complex antigens to
MHC-restricted, antigen-specific T lymphocytes (Del Pozo et al., 1992; Weller et al.,
1993). Furthermore, eosinophils express the costimulatory molecules required for
effective antigen presentation (Tamura et al., 1996), and ligation of costimulatory
molecules on the eosinophil cell surface can induce the release of eosinophil derived
cytokines (Woerly et al., 1999; Woerly et al., 2002). Therefore the eosinophil may also
regulate immune responses.¶
To date, no studies have demonstrated the ability of eosinophils to modulate activated T
lymphocyte function via presentation of relevant antigen in the context of MHC class II
(MHC-II), concomitant with Th2 cytokine release. In the experiments described in this
thesis, murine eosinophils have been observed to rapidly migrate to sites of antigen
deposition within the airways mucosa of naïve mice, suggesting a potential role for this
granulocyte in the primary response to inhaled antigen. However, human allergic
diseases are often diagnosed after the establishment of allergic responses, and symptom
development. Therefore, a murine model of allergic airways disease (AAD) was used to
investigate the ability for eosinophils to participate as antigen presenting cells (APCs),
and thereby modulate activated T lymphocyte function both in vitro and in vivo.
Detailed histological analysis of the pulmonary draining lymph nodes following antigen
challenge in sensitised mice revealed a rapid infiltration of eosinophils into this tissue,
which preceded the accumulation of eosinophils in bronchoalveolar lavage fluid
(BALF). This suggested that eosinophils were preferentially translocating to the
draining lymph nodes following antigen challenge, and that the subsequent
accumulation of these cells in the BALF was a consequence of continued antigen
delivery to the lower airways.¶
Eosinophil trafficking to lymphoid tissue via the afferent lymphatics was substantiated
using electron microscopy of lymph node sections and the intravenous (i.v.) transfer of
fluorescently labeled eosinophils, which did not traffic to lymph nodes via the blood.
During the resolution of AAD, eosinophils were noted for their persistence in the
pulmonary draining lymph nodes. These observations suggested a continued modulation
of T cell function by lymph node dwelling eosinophils during AAD resolution,
particularly in light of recent observations for draining lymph node T cell proliferation
following instillation of antigen-pulsed eosinophils into the allergic mouse lung (Shi et
al., 2000).¶
To further investigate the antigen presenting capacity, eosinophils were obtained from
the BALF of mice with AAD, and their surface expression of MHC class II (MHC-II)
proteins and costimulatory molecules confirmed using flow cytometric analysis. The
ability to acquire and process complex antigen both in vitro and in vivo was also
confirmed using naturally quenching fluorescenated ovalbumin (OVA), which is
degraded into fluorescent peptides by the action of intracellular proteases. Thus,
eosinophil expression of the surface molecules necessary for effective antigen
presentation was confirmed, as was their ability to process complex antigen. Further
investigations revealed that eosinophils can present complex OVA antigen to CD4+ T
lymphocytes obtained from the allergic mouse, and to in vitro derived OVA-specific
Th2 cells. In the presence of exogenous antigen, eosinophils co-cultured with T
lymphocytes were able to induce Th2 cytokine production, and demonstrated an ability
for eosinophils to modulate T lymphocyte function in vitro.¶
The ability for eosinophils to act as antigen presenting cells in vivo was also
investigated. Eosinophils obtained from the antigen-saturated lungs of OVA sensitised
and challenged mice were transferred to the peritoneal cavities of naïve host mice.
When subsequently challenged with aerosolised OVA, eosinophil recipients developed
a pulmonary eosinophilia similar to that of OVA sensitised and challenged mice. To
validate this finding, the experimental procedure was altered to accommodate the use of
non-allergy derived eosinophils, which were pulsed with OVA in vitro, prior to transfer
into naïve recipients. When subsequently challenged with aerosolised OVA, eosinophil
recipients developed a peripheral blood and pulmonary eosinophilia, and stimulation
with OVA induced IL-5 and IL-13 cytokine production from pulmonary draining lymph
node cells. Notably, the AAD induced by transfer of antigen pulsed eosinophils did not
induce detectable OVA-specific IgG1, which may be attributed to the lack of soluble
antigen required for B cell antibody production.¶
During the course of these investigations, an OVA T cell receptor (TCR) transgenic
mouse (OT-II) was procured with a view to defining the interaction between eosinophils
and activated T lymphocytes (Barnden et al., 1998). Despite having specificity for the
OVA323-339 peptide, an immunodominant epitope that skews naïve T cell responses
towards Th2 cytokine release (Janssen et al., 2000), T lymphocytes from the OT-II
mouse preferentially secreted IFN-γ in response to stimulation with either OVA peptide
or OVA. These mice were further characterised in a mouse model of AAD, and found to
be refractory to disease induction and progression, which may be attributed to
significant IFN-γ secretion by transgenic CD4+ T lymphocytes during antigen
sensitisation. Indeed, these cells were noted for their ability to attenuate pulmonary
eosinophilia when transferred to OVA sensitised and challenged wild type mice,
although serum OVA-specific IgG1, peripheral blood eosinophilia levels and airways
response to methacholine challenge remained intact.¶
Knowledge of the biased Th1 phenotype in naïve OT-II provided a unique opportunity
to investigate the fate of T lymphocytes bearing high affinity OVA-specific TCRs
following neonatal antigen exposure to soluble OVA. In a previous study, subcutaneous
(s.c.) administration of soluble OVA to wild type neonatal mice was suspected to have
deleted OVA-specific T cells from the T cell repertoire (Hogan et al., 1998a). Using
flow cytometry and TCR specific antibody, the delivery of s.c. OVA to OT-II neonates did not alter transgenic T cell populations in adult mice. Instead, it was surprising to
find a skewing towards the Th2 phenotype and loss of IFN-γ secretion following OVA
sensitisation and challenge in adult mice. A mechanism for this reprogramming of the
transgenic T cell from the Th1 to a Th2 phenotype following OT-II neonatal exposure
to soluble OVA is proposed, and further experimentation may validate this hypothesis.¶
In conclusion, eosinophils residing in the allergic lung have the capacity to interact with
activated T cells, both within this tissue and the draining lymph nodes. Despite their
relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils
may participate en masse in the serial triggering of activated TCRs, and provide
appropriate costimulatory signals that modulate T lymphocyte function. Through the
elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting
eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases.
Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via
degranulation, and such activity has recently been observed in a parasite model (Shinkai
et al., 2002). Finally, experiments in the OT-II mouse have provided valuable
information to suggest that therapies designed to modulate eosinophil numbers in
allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of
limited benefit. The results shown here suggest that airways dysfunction remains intact
despite significantly reduced pulmonary eosinophilia
| Identifer | oai:union.ndltd.org:ADTP/216799 |
| Date | January 2004 |
| Creators | MacKenzie, Jason Roderick, Jason.Mackenzie@ipaustralia.gov.au |
| Publisher | The Australian National University. The John Curtin School of Medical Research |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.anu.edu.au/legal/copyrit.html), Copyright Jason Roderick MacKenzie |
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