In addition to their role as pro-inflammatory cells in allergy, asthma eosinophils are also involved in certain repair and fibrotic processes consequent to allergic inflammation. For example, in the skin of atopic subjects, eosinophil-derived fibrogenic factors (transforming growth factor-
(TGF-1) and interleukin (IL)-13) were temporally associated with fibroblast-associated tenascin and procollagen-I immunoreactivity, and the formation of 
-smooth muscle (SM) actin+myofibroblasts, following local allergen challenge (Phipps et al, 2002). In addition the specific reduction of eosinophils in asthmatic airways at baseline by intravenous infusions of an anti-IL-5 monoclonal antibody decreased the deposition of the extracellular matrix (ECM) proteins, tenascin, lumican, and procollagen III, within the reticular basement membrane (Flood-Page et al, 2003b).
IL-5 is essential for the terminal differentiation of the committed eosinophil precursor (Sanderson, 1992). It is also involved in eosinophil migration and priming (Sehmi et al, 1992) and prolongs the survival of the cell in tissues (Rothenberg et al, 1989). More recently monoclonal antibodies against IL-5 have been prepared and administered as a single intravenous infusion to both mild atopic (Leckie et al, 2000), as well as chronic, severe asthmatics (Kips et al, 2003). These have produced no appreciable effects on either the late asthmatic reaction, airway hyperresponsiveness or other clinical outcomes including lung function. However, although anti-IL-5 almost totally ablated eosinophils in the blood and sputum (Leckie et al, 2000), tissue eosinophils were reduced rather than depleted (Flood-Page et al, 2003a), possibly as a result of downregulated IL-5R expression of airway eosinophils (Liu et al, 2002;Gregory et al, 2003).
In this study, we have measured eosinophils, tenascin, and the size of the late-phase reaction (LPR) in allergen-challenged skin sites before and after anti-IL-5. The cutaneous LPR, elicited in atopic subjects, is characterized by an edematous, red, and slightly indurated swelling that peaks 6–9 h after intradermal allergen challenge. The LPR is often considered as a model of allergic inflammation since it is associated with local infiltration by various cell types including eosinophils, basophils, and neutrophils. The eosinophil in particular has been considered as an important effector cell in producing the macroscopic appearance of the LPR possibly through the release of lipid mediators such as cysteinyl leukotrienes (Reshef et al, 1989;Zweiman et al, 1991;Wardlaw et al, 1995). Although increases in the size of the LPR accompanies increases in the numbers of eosinophils up to 6 h after allergen injection, thereafter the cell persists in tissues whereas the LPR declines fairly rapidly (Phipps et al, 2002). For this reason we were interested to study the effect of reduction or depletion of eosinophils, by anti-IL-5, on the magnitude of the allergen-induced LPR.
In this study, we performed skin biopsies 6 and 48 h after cutaneous allergen challenge (from the same patients described as previously byFlood-Page et al, 2003b) before and after the infusion of an anti-IL-5 monoclonal antibody. We chose the 6 h time point as optimal for studying the effect of eosinophil depletion on the LPR since in time-course studies up to 72 h this was shown to be the peak of the characteristic redness and swelling (Phipps et al, 2002). A 48 h biopsy time point was chosen to enable us to determine whether this procedure affected the later expression of a marker of repair (as previously shown for tenascin) (Phipps et al, 2002).
Results
Anti-IL-5 reduces eosinophil infiltration but does not effect the size of the cutaneous LPR
Figure 1 shows the skin Congo red+eosinophil counts of biopsies taken from the diluent- and allergen-injected sites in subjects receiving anti-IL-5 or placebo. At 6 h after intradermal allergen challenge there was no significant difference (pre vs post) in the numbers of skin eosinophils in subjects receiving placebo; however, the counts were significantly less (pre vs post, p=0.002) in those receiving intravenous anti-IL-5 monoclonal antibody, with a between-group difference of p=0.0015. This represented a median change in eosinophil numbers at 6 h of -83% for anti-IL-5 and +33% for placebo. At 48 h after intradermal allergen challenge, there was no significant difference (pre vs post) in the numbers of skin eosinophils in the placebo group but the counts were significantly less (pre vs post, p=0.003) in those receiving anti-IL-5. The between-group difference at 48 h was p=0.0025. The mean eosinophil counts of all the diluent-injected sites was less than 2 cells per mm2. Similar results were obtained with anti-MBP with a between-group difference at 6 h (anti-IL-5 vs placebo) of p=0.01 (data not shown). The number of degranulating cells was also counted Figure 2. At 6 h after intradermal allergen challenge there was no significant difference (pre vs post) in the numbers of degranulating eosinophils in subjects receiving placebo; however, the counts were significantly less (pre vs post, p=0.001) in those receiving intravenous anti-IL-5 monoclonal antibody, with a between-group difference of p=0.029. At 48 h after intradermal allergen challenge, there was no significant difference (pre vs post) in the numbers of degranulating skin eosinophils in the placebo group but the counts were significantly less (pre vs post, p=0.0117) in those receiving anti-IL-5.
Figure 1.
Effect of anti-IL-5 mAb on allergen-induced eosinophil infiltration. Eosinophil numbers are expressed as the number of Congo red+cells (mean
SEM) per square millimeter of skin biopsy (n=11–13). There was no significant between-group difference (anti-IL-5 vs placebo) in the allergen-induced increases in eosinophils before treatment at either 6 or 48 h (P=0.1858 and 0.31, respectively). The differences between pre- and post-treatment (active or placebo) were analyzed by Wilcoxon signed-rank test. The Mann–Whitney U test was used for intergroup comparison. IL, interleukin; mAb, monoclonal antibody.
Figure 2.
Effect of anti-IL-5 mAb on the numbers of MBP+degranulating eosinophils after allergen-challenge. Degranulating MBP+eosinophil numbers are expressed as the number of cells (meanSEM) per square millimeter of skin biopsy (n=11–13). The differences between pre- and post-treatment (active or placebo) were analyzed by Wilcoxon signed-rank test. The Mann–Whitney U test was used for intergroup comparison. IL, interleukin; mAb, monoclonal antibody; MBP, major basic protein.
As previously shown (Ying et al, 1999), there were also significant allergen-induced increases in BB1+basophils, elastase+neutrophils and CD4+T cells at both time points but their numbers were unaffected by infusions of anti-IL-5 (data not shown).
In contrast, administration of anti-IL-5 (or placebo) had no significant effect on the 6 h late-phase cutaneous reaction Figure 3. The magnitude of the 6 h LPR was similar to that previously observed (Frew and Kay, 1988;Ying et al, 1999;Phipps et al, 2002). In the majority of subjects, in both the active and placebo group, the 48 h LPR had, with the exception of a few individuals, largely resolved. But in five individuals who had a small waning 48 h reaction, anti-IL-5 appeared to have a small, but NS, inhibitory effect (individual data not shown).
Figure 3.
Effect of anti-IL-5 mAb on the size of the allergen-induced cutaneous LPR. Data represent meanSEM; n=11–13. The differences between pre- and post-treatment (active or placebo) were analyzed by Wilcoxon signed-rank test. The Mann–Whitney U test was used for intergroup comparison. IL, interleukin; mAb, monoclonal antibody; LPR, late-phase reaction.
Anti-IL-5 deplete tenascin-positive cells in 48 h biopsies
Figure 4 shows the numbers of tenascin+fibroblast-like cells from biopsies taken from diluent-and allergen-injected sites pre- and post-treatment. Tenascin+cells were predominantly located in the lower dermis and were identified morphologically as fibroblasts, appearing fusiformic in shape with elongated nuclei. There were negligible numbers of tenascin+cells (mean <1 cell per mm2) at diluent-injected sites. At 6 h after intradermal allergen challenge there was no significant difference (pre-placebo vs post-placebo, or pre-anti-IL-5 vs post-anti-IL-5) in the numbers of fibroblast-like tenascin+cells. The between-group difference at 6 h was also NS. At 48 h, however, the counts were significantly less in those receiving intravenous anti-IL-5 monoclonal antibody (pre vs post, p=0.003), but not in those receiving placebo. The between-group difference at 48 h was also significant (p=0.0256). There was also a highly significant correlation between the 
change in eosinophils and the change in tenascin+cells in those receiving anti-IL-5 (p=0.0005; Figure 5). Examples of Congo red+eosinophils and immunostaining for tenascin+cells, pre- and post-anti-IL-5, are shown in Figure 6.
Figure 4.
Effect of anti-IL-5 mAb on allergen-induced tenascin expression. The results are expressed as the number of tenascin+cells (meanSEM) per square millimeter of skin biopsy (n=11–13). There were no significant between-group differences (anti-IL-5 vs placebo) in the allergen-induced increases in tenascin+cells before treatment at either 6 or 48 h (P=0.3402 and 0.1315, respectively). The differences between pre- and post-treatment (active or placebo) were analyzed by Wilcoxon signed-rank test. The Mann–Whitney U test was used for intergroup comparison. IL, interleukin; mAb, monoclonal antibody.
Figure 5.
Correlation between the change in tissue eosinophils and change in tenascin+cells after allergen challenge. The results are expressed as the change in the number of eosinophils and the change in the number of tenascin+cells (meanSEM) per square millimeter of skin biopsy (n=11), following active treatment at the 48 h time point. Correlations were obtained by Spearman's method with correction for tied values.
Figure 6.
Effect of anti-IL-5 mAb on tissue eosinophils and tenascin+cells Examples of Congo red+eosinophils and immunostaining for tenascin+cells, pre- and post-anti-IL-5 (magnification 
200).
Discussion
This study shows that selective reduction of tissue eosinophils (as opposed to blood and sputum eosinophils) does not significantly affect the magnitude of the allergen-induced LPR. Although eosinophil accumulation was not completely abrogated by anti-IL-5, these data suggest, nevertheless, that eosinophils are unlikely to be essential for the development of the LPR at its peak (i.e. 6 h). It was previously shown that administration of anti-IL-5 had no effect on the late asthmatic reaction or airway hyperresponsiveness (Leckie et al, 2000) although in that report it was not ascertained whether the intervention actually depleted eosinophils in the relevant tissue, i.e. the bronchial mucosa. Indeed, it appears that whereas on the one hand anti-IL-5 will prevent the allergen-induced increase in eosinophils in the skin of (otherwise normal) atopics Figure 1 (where there are virtually no eosinophils at baseline), this treatment, even when administered on several occasions, over several weeks, had only a partial effect (median depletion of 55%) on baseline eosinophils in the bronchial mucosa (Flood-Page et al, 2003a), which in asthmatics is already mildly inflamed and contains appreciable numbers of eosinophils even when "unprovoked" (Azzawi et al, 1990).
The precise mechanisms involved in the redness, swelling, and slight induration that characterizes the peak (6–9 h) late-phase skin response remains unclear. Although this study indicates that eosinophils do not appear to be essential, other cell types such as the basophil may be involved. Basophils are a rich source of mediators including histamine and cysteinyl leukotrienes (Church et al, 1997;Macfarlane et al, 2000) and respond to allergen via IgE bound to Fc
R1. Furthermore, the peak of the late-phase response is usually between 6 and 24 h, at which time basophil numbers are maximal at skin sites after allergen challenge (Ying et al, 1999). Although IL-5 is also believed to act as a terminal basophil differentiation factor (Denburg et al, 1991), however, anti-IL-5 did not appear to affect the numbers of infiltrating BB1+basophils as identified either in 6 or 48 h skin biopsies (data not shown).
We were able to study the effects of anti-IL-5 on the LPR at both the 6 and 48 h time points. It was previously shown that the cutaneous LPR plateaus at 6–9 h whereas the peak of eosinophils is variable between 6 and 24 h (Phipps et al, 2002). Thus the 6 h time point was optimal for studying the relationship between eosinophils and the LPR, and as previously shown, tenascin+cells peaked at 48 h (Phipps et al, 2002). The findings presented here are in agreement with our previous observations of a dissociation between eosinophil numbers and the size of the cutaneous reaction (Phipps et al, 2002). Thus, whereas increases in eosinophil numbers and the size of the LPR increased in parallel at the 1, 3, 6, and 24 h time points, thereafter eosinophils persist in the tissue whereas the LPR rapidly resolves. In previous studies, we showed that tissue eosinophils persisted for up to 7 d but that the LPR was usually absent at 72 h (Ying et al, 1999). Although the 6 h LPR was not affected by anti-IL-5, five of the 11 individuals who had a small positive 48 h LPR before anti-IL-5 treatment showed a reduction in their LPR after treatment Figure 3. Therefore, we cannot totally exclude a role for the eosinophil at later time points, however, classically the LPR refers to events observable between 6 and 9 h.
A previous publication showed that eosinophil granule protein deposition was prominent in the cutaneous LPR in atopics, peaking between 4 and 8 h and persisting for 48 h (Leiferman et al, 1990). Our study largely confirms these findings. Degranulating MBP+cells were observed in almost equal numbers at 6 and 48 h in the placebo group (pre and post) Figure 2. After anti-IL-5 there were very few degranulating cells at either time point. Furthermore, the LPR is transient and wanes after 6–9 h (Phipps et al, 2002) whereas, as stated, eosinophils and eosinophil products persist for many hours and days. This makes it unlikely that degranulating eosinophil products were contributing to the 6 h LPR although in some subjects there may be a small eosinophil component to the waning 48 h response. It is also noteworthy that, although eosinophil-derived basic proteins are toxic for various cell types, including epithelial cells, there is no evidence that they cause the vasodilation and edema which characterizes the late-phase skin response.
Anti-IL-5 had a slightly more marked effect on the number of Congo red+eosinophils than MBP+cells. But basophils also contain small amounts of MBP (Ackerman et al, 1983).
Our second novel finding was the demonstration that reduction of the allergen-induced eosinophil response abrogated the 48 h increase in tenascin+fibroblast-like cells Figure 4. Furthermore, there was a correlation between the decrease in the numbers of eosinophils and the decrease in the numbers of tenascin+cells Figure 5 and so supports the view that upregulation of tenascin expression by fibroblast-like cells following intradermal allergen challenge is partly under the control of infiltrating eosinophils presumably through the release of fibrogenic factors such as TGF-. This is in keeping with our recent study showing, in these same patients, decreased deposition of the ECM proteins, tenascin, lumican, and to a lesser extent, procollagen III, within the reticular basement membrane, together with decreased numbers of TGF-+eosinophils in the bronchial mucosa, after anti-IL-5 treatment (Flood-Page et al, 2003b). Due to limitations of available tissue, we were unable to confirm that the numbers of TGF-+eosinophils were similarly decreased in the skin. Although, in our previous study in the skin (Phipps et al, 2002) we had shown that allergen induces the upregulation of other markers of remodelling (i.e. -SM actin+myofibroblasts and procollagen-I+cells), we were only able to show, in this study, an effect of anti-IL-5 on tenascin+cells. There are likely to be many other sources of TGF- and other fibrogenic growth factors (such as macrophages, T cells and neutrophils), however, which would not be affected by the infusions of anti-IL-5. Thus, the precise contribution of eosinophils (as compared to other cell types) to repair and remodelling processes must await further investigation.
Tenascin is a highly regulated member of the matricellular family that is expressed during development, growth, and in response to injury (Erickson and Bourdon 1989;Ruegg et al, 1989). In asthmatic airways the balance of the ECM proteins is altered and the deposition of tenascin, together with several other ECM proteins, is increased (Laitinen et al, 1997). The ECM not only forms a network of molecules that support the airways, it is also a dynamic network that has the capacity to influence cellular function. Tenascin has been demonstrated to act as a permissive substance that can prevent or allow cell migration (Treasurywala and Berens, 1998) and we have previously shown a significant upregulation of tenascin expression within fibroblast-like cells, at the vascular SM basement membrane and in and around bundles of SM in response to intradermal allergen challenge (Phipps et al, 2002). It is possible that tenascin, in conjunction with other proteins, facilitates cellular migration through the interstitial matrix towards sites of tissue injury and thus may play a role in remodelling.
The late-phase skin reaction is often regarded as a model of atopic dermatitis because in both situations the histopathological picture is of an eosinophilic cell mediated hypersensitivity reaction. In atopic dermatitis, however, there is relatively little deposition of collagen and ECM proteins. Thus a single allergen challenge in otherwise healthy normal skin is unlikely to lead to marked remodelling (Ying et al, 1999;Phipps et al, 2002). Nevertheless, it is associated with type I collagen deposition and expression of the pro-fibrotic cytokines IL-11 and IL-17 (Toda et al, 2003) and, as such, the late-phase skin reaction could serve as a general model for events associated with the laying down of collagen and other ECM proteins, not only in skin disease but also in asthma. In the airways of asthmatics, for instance, cells of the epithelial–mesenchymal trophic unit are now recognized as active participants in the inflammatory process (Holgate et al, 2000). Although this remodelled phenotype is generally believed to be the result of chronic inflammation, our recent findings in the skin (Phipps et al, 2002) and those ofGizycki et al (1997) in the airways suggest that tissue remodelling is an acute/subacute process, resulting from allergen-induced interactions between eosinophils and other inflammatory cells with mesenchymal cells.
In a mouse model of atopic asthma,Blyth et al (2000) demonstrated a reduction in the development of subepithelial reticular basement membrane thickness by treatment with anti-IL-5 at the time of allergen challenge. In agreement with the reported effects of anti-IL-5 on subepithelial basement membrane thickening in the airways (Flood-Page et al, 2003b), we demonstrated that the selective depletion of eosinophils in response to intradermal challenge led to a significant reduction in the numbers of tenascin+cells at 48 h. Although in a hamster model of incisional wound healing depletion of eosinophils with anti-IL-5 accelerated the rate of wound closure by re-epithelialization (Yang et al, 1997), this study may simply reflect the pleiotropic activity of TGF- on different components of the healing response. Smad-3 (a downstream signal transducer for TGF-) heterozygous mice also demonstrate accelerated re-epithelialization compared with wild-type mice (Shipley et al, 1986;Ashcroft et al, 1999), an effect mediated by removal of the inhibitory action of TGF- on keratinocyte chemotaxis and proliferation (Sehmi et al, 1992). Although together these studies suggest that eosinophils, in response to injury, signal to and activate epithelial and mesenchymal cells, it remains to be determined whether eosinophil-induced tissue remodelling is beneficial or detrimental to the host.
The observed effect of anti-IL-5 on tenascin may have been via a direct effect on fibroblasts, or other mesenchymal cells, rather than through a reduction of tissue eosinophils; however, there are no data reporting enhanced synthesis of ECM protein by IL-5 and fibroblasts do not appear to express the IL-5R chain (T.-T. Ou and S. Phipps, unpublished). Furthermore, there was a direct correlation between the changes in eosinophils and changes in tenascin+cells in those receiving anti-IL-5 Figure 5.
In conclusion our data suggest that eosinophils are unlikely to be essential for the swelling and induration that characterizes the late-phase skin reaction at its peak (i.e. 6–9 h) and that they may play a role in remodelling processes associated with allergic inflammation.
Materials and Methods
Volunteers for anti-IL-5 study
The study was approved by the ethics committees of the Royal Brompton and Harefield NHS Trust and the London Chest Hospital, and was performed in accordance with the guidelines of the Declaration of Helsinki. All volunteers gave informed consent prior to participation in the study. 24 volunteers were recruited with a history of mild asthma, with an forced expiratory volume in 1 s (FEV1) of 
70% of the normal value for age and height and within an 18–55 y age range. All volunteers were atopic defined by a positive skin prick test to one or more aeroallergens (Dermatophagoides pteronyssinus, cat, dog, and mixed grass and tree pollen (all ALK, Horsholm, Denmark)) and were well controlled with short-acting 2 agonist alone, had no history of worsening asthma or upper respiratory tract infection in the preceding 4 wk, and had not taken inhaled or oral corticosteroids, or other anti-inflammatory drugs or anti-histamines in the preceding 8 wk. There was documented airway hyperresponsiveness as shown by a provocative concentration causing a 20% fall in FEV1 to histamine of 
4.0 mg per mL. All volunteers were non-smokers for at least the preceding 6 mo with no more than a 10-pack year lifetime smoking history.
Study design and processing of specimens
This was a two-center double-blind, placebo-controlled, parallel group study designed to evaluate the effects of an anti-IL-5 monoclonal antibody on baseline bronchial mucosal and bone marrow eosinophils and allergen-induced skin eosinophils. The results of studies on the effect of treatment on blood, bronchial, and bone marrow eosinophils has been reported elsewhere (Flood-Page et al, 2003a,b). Volunteers were randomized to receive either mepolizumab (750 mg) or placebo, administered as an intravenous infusion over 30 min. The second and third infusions of the study drug were given 4 and 8 wk after the first infusion. Skin biopsies were obtained 2 d before the first infusion of study medication and between 1 and 2 wk after the third infusion. All injections were performed with a 29-gauge needle and a 0.5 mL plastic syringe. Using this method, 30 biological units of either grass, house dust mite, cat, or dog allergen was injected intradermally into two sites on the extensor aspect of the forearms of each subject. The size of the cutaneous reaction was determined at 6 and 48 h by measuring resistance to the movement of a sharpened pencil and expressed as the mean diameter (mm) as previously described (Ying et al, 1999). There were no appreciable differences in the size of the late-phase skin responses between the various allergens used. An additional site was injected with a similar volume of diluent. Macroscopic responses were measured at 6 and 48 h and permanent sticky tape records of the outlines of the responses made. A 4 mm disposable biopsy punch was used to take a biopsy from the center of the reaction at 6 and 48 h after using 1% plain lignocaine for local anesthesia. The control site injected with diluent was biopsied at 6 h. In this way, each patient served as his/her control. Tissue biopsies were immediately fixed in 4% paraformaldehyde and washed in 15% PBS-buffered sucrose (Sigma, Poole, UK), embedded in OCT (optimal cutting temperature), then snap-frozen in isopentane precooled in liquid nitrogen. Cryostat sections (<8 
m) were cut from biopsies, mounted onto Superfrost Plus slides, dried overnight at 37°C, then stored with silica gel at -80°C until use (all VWR, Dagenham, UK unless stated).
Histochemistry and immunohistochemistry
Eosinophil accumulation was determined by Congo red, a selective stain for eosinophils in tissue sections from the skin as described previously (Ying et al, 2002). Briefly, sections were washed in PBS for 5 min then incubated in 0.5% Congo red (Sigma) in ethanol/0.1 M glycine (1:1) for 5 min at room temperature. The slides were then rinsed in 70% ethanol until the background became clear, then mounted in glycergel (Dako, Cambridge, UK). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique was used to enumerate cells immunoreactive to a monoclonal antibody (mAb) against CD4, eosinophil major basic protein (MBP), neutrophil elastase, basophil BB1 (a generous gift from Dr A. Walls, University of Southampton, UK), and tenascin (Caltag-Medsystems, Towcester, UK). The APAAP technique, with source of reagents, was performed as described previously (Ying et al, 1999). Tissue sections were developed with fast red (Sigma) as chromogen for signal visualization (Dako). Cells were counter stained with Harris' Hematoxylin (VWR) and mounted in glycergel. Positive cells stained red after development with fast red. Substitution of the primary antibody with an irrelevant isotype-matched antibody of the same species was used as a negative control. One biopsy specimen from each time point was evaluated from each patient.
Quantitation and statistical analysis
Slides were encoded and counted in a blinded fashion using an Olympus microscope (Olympus Optical Co., London, UK). The whole section was counted and the total number of single positive cells expressed as cell per square millimeter of biopsy. Data were analyzed using a statistical software package (Minitab Release 13.1, Minitab, State College, Pennsylvania). Non-parametric statistics were used throughout the study. The Wilcoxon signed-rank test was used to analyze intragroup changes in the numbers of immunoreactive-positive cells in response to allergen. The Mann–Whitney U test was used for intergroup comparisons. Correlation coefficients were obtained by Spearman's rank-order method. A p-value of >0.05 was accepted as non-significant (NS).
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