To further identify the migration pathway of eosinophils to pulmonary compartments during allergen provocation, 2 x 106 fluorescently labeled eosinophils from IL-5 transgenic mice were injected i.v. into allergic wild type mice following the third aeroallergen challenge. Within 2 hours of transfer, labeled cells were detected in the lung, spleen, and blood, but not in BALF, PBLN tissue, nor the gut (Figures 3.6 and 3.7). By 20 hours, labeled eosinophils were still migrating into the lung tissue, and fluorescent cells were still undetected within PBLN tissue (not shown). These results suggest that eosinophils do not passage directly via the blood and high endothelial venules to PBLN following allergen provocation, but migrate via the pulmonary parenchyma and draining lymphatics.
Chapter 3 Analysis Of Tissue Eosinophilia During The Progression And Resolution Of Allergic Airways Disease
Figure 3.6. Trafficking of labeled eosinophils to pulmonary and lymphoid tissue following allergen provocation. Within 2 hours of i.v. transfer, labeled cells were detected in spleen, lung and blood, but not in PBLN tissue. Region 1 (R1) represents the area in which eosinophil would fall based on forwards (FSC-Height) and side scatter (SSC-Height). Detection of H33342 fluorescence (F32-Height) for each tissue is given below each window, and is gated for cells within R1 only. Fluorescent cells are represented in the upper left quadrant.
Chapter 3 Analysis Of Tissue Eosinophilia During The Progression And Resolution Of Allergic Airways Disease
Figure 3.7. Trafficking of labeled eosinophils to pulmonary and lymphoid tissue following allergen provocation. Within 2 hours of i.v. transfer, labeled cells were not detected in BALF nor the small intestines (GUT). Region 1 (R1) represents the area in which eosinophil would fall based on forwards (FSC-Height) and side scatter (SSC-Height). Detection of H33342 fluorescence (F32-Height) for each tissue is given below each window, and is gated for cells within R1 only. Fluorescent cells are represented in the upper left quadrant. Eosinophils stained with H33342 dye prior to transfer (H33342) are shown in comparison to unstained cells (NEG CONTROL).
3.4 DISCUSSION
In the experiments described in Chapter 2, it was shown that antigen deposition in the naïve lungs of PMBS-treated mice could induce eosinophil accumulation in the airway lumen via an eotaxin dependent mechanism. Eosinophils are prominent features in allergic mucosal surfaces of the lung and gastrointestinal tract (Gleich et al., 1993; Rothenberg, 1998), and these cells are currently thought to act as effector cells, that induce pathological changes through the release of granular proteins and
proinflammatory mediators. The degranulation status of mouse eosinophils in allergic airways disease models is currently under intense scrutiny (Stelts et al., 1998; Persson and Erjefalt, 1999; Denzler et al., 2001; Malm-Erjefalt et al., 2001), however the results presented in this Chapter do not attempt to delineate a pathological role for eosinophils. Rather, attention has been focused on other potential roles for eosinophils in the
regulation of allergic airways disease through characterisation of their spatial and temporal distribution during the development and resolution of allergic inflammation of the lung.
Detailed analysis of eosinophil trafficking to the lung and draining lymphoid tissue during both the progression and resolution of allergic airways disease has provided a valuable insight into the potential role eosinophils may play in disease regulation. In this Chapter, eosinophils were observed to traffic to sites of antigen deposition (airways lumen) and T cell expansion (PBLN) in response to antigen provocation of the airways. An increase in peripheral blood eosinophils was observed in as little as 6 hours
following antigen provocation (Figure 3.2a), and a significant increase in eosinophils in the airways lumen was observed within 48 hours (Figure 3.2b). These results agree with those of Ohkawara et al., who have also used a mouse model of allergic airways disease to measure the kinetics of eosinophil migration into pulmonary tissues (Ohkawara et al., 1997). In their model, Ohkawara et al. observed eosinophil adhesion to pulmonary endothelial cells in as little as 3 hours following airways challenge, which paralleled an increase in peripheral blood eosinophilia and subsequently led to infiltration of the airways lumen within 24 hours.
Antigen specific lymphocytes circulating through the allergic mouse lung can quickly respond to relevant antigen deposition through rapid synthesis and secretion of IL-4, IL- 5 and IL-13 (Gajewska et al., 2001), which act in concert to promote tissue
eosinophilia. In this Chapter, a significant increase in the numbers of eosinophils residing in the draining PBLN was noted 3 hours following the initial exposure to antigen, and preceded the accumulation of eosinophils in the BALF (Figures 3.2b and c). It is tempting to speculate that eosinophils may preferentially translocate to the lymph node during the early phase of airways inflammation, and that repeated exposure to antigen then promotes the accumulation of eosinophils in the lung and BALF. This effect may be seen in Figures 3.2b and c, wherein eosinophils accumulate in both draining PBLN and the airways lumen following repeated exposure to OVA on days 25 and 26 (i.e. at 48 and 72 hours following the initial exposure).
Following the cessation of antigen provocation, airways eosinophils become apoptotic and may be engulfed by airway macrophages (Kodama et al., 1998), or small airway epithelial cells (Walsh et al., 1999). This may be directly related to the loss of antigen induced inflammatory cytokines such as IL-5, IL-3 and GM-CSF (Stern et al., 1992; Simon and Blaser, 1995), and suggests a mechanism whereby eosinophils are removed from lung tissue following the cessation of antigen provocation. In this Chapter, peripheral blood eosinophil levels and the numbers of eosinophils in BALF returned to baseline within 12 and 15 days, respectively, following the cessation of airways
challenge with OVA (Figures 3.5a and b). Notably, the numbers of eosinophils residing in lung tissue remained elevated until the pool of eosinophils in the airway lumen were depleted (Figures 3.5b and c). These results correspond to the observations made by Ohkawara et al., who in a similar model, have noted resolution of BALF and blood eosinophil levels at 15 and 21 days, respectively, and a return to a normal bronchial epithelium by day 21 (Ohkawara et al., 1997).
Interestingly, PBLN eosinophil numbers continued to increase until day 6 following the cessation of antigen challenge, before falling in parallel with lung tissue levels over the next 12 days and finally to baseline levels by day 18 (Figure 3.5c). Mouse eosinophils traffic to the draining lymph nodes during the recovery phase of helminth infection (Friend et al., 2000), and in conjunction with data presented in this Chapter, suggests a potential role for eosinophils in the regulation of draining lymph node function during the resolution of Th2 cytokine driven diseases. In the rat, peripheral lymph node high endothelial cells express VCAM-1, the expression of which is upregulated by cytokines (May et al., 1993). Antibodies to the cognate receptor VLA-4 have been shown to reduce eosinophil recruitment to tissue in vivo (Weg et al., 1993), and thus identifies a
possible route of entry for peripheral blood eosinophils into the lymph node via high endothelial venules. In this Chapter, fluorescently labeled eosinophils were not observed to enter draining thoracic lymph nodes via the blood (Figures 3.6 and 3.7), suggesting that the afferent lymphatics are the major entry point for eosinophils found to reside in peribronchial lymphoid tissue following antigen challenge of the airways.
Eosinophils are phagocytic cells with antigen presenting capacity (Del Pozo et al., 1992; Weller et al., 1993), and the ability of eosinophils situated in the airways lumen to migrate via the lymphatics to sites of antigen presentation has recently been
confirmed (Shi et al., 2000). Thus, eosinophils that are recruited to the antigen challenged lung in sensitised mice may then sample the extracellular milieu, and phagocytose endogenous antigen. Within inflamed lung tissue, eosinophils may then process and present endogenous antigen to activated memory T lymphocytes, and thereby modulate local T cell function and disease progression/resolution. Alternatively, recruited eosinophils may sample the contents of the airway lumen, before translocation back across airways epithelium and subsequent trafficking to the draining lymph nodes. In these tissues, eosinophils may reside for prolonged periods of time following antigen challenge in the sensitised organism (Figure 3.5c), and may actively present airways derived antigens to antigen-specific T lymphocytes. Thus, the numerous eosinophils that are recruited to the antigen challenged lung in the sensitised organism are well placed to modulate T lymphocyte function within local tissues and distally via the draining lymphatics. It is interesting to note that low numbers of eosinophils were present in the blood, airways lumen, and draining lymphoid tissue prior to antigen inhalation (Figure 3.2, zero time points), which suggests a role for baseline eosinophils in immunological responses associated with immune surveillance and potentially, T cell expansion.
It would be interesting to assess the surface expression of MHC-II and associated costimulatory molecules, and to further investigate any antigen presenting function attributable to lung eosinophils. This may further our understanding of the potential for eosinophil/T cell interactions wherein eosinophils may regulate T cell function during chronic antigen exposure to the lung.