Effect of Interferon- on Allergic Airway Responses in Interferon-deficient Mice

Makoto Yoshida, Richard Leigh, Koichiro Matsumoto, Jennifer Wattie, Russ Ellis, Paul M. OByrne, and Mark D. Inman
Firestone Institute for Respiratory Health, Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Interferon (IFN)- reduces airway responses after allergen challenge in mice. The mechanisms of this effect are not clear. These studies investigate whether IFN- can reverse prolonged airway responses after allergen challenge in IFN-deficient (IFN-KO) mice. Sensitized mice (IFN-KO and wild-type [WT]) were challenged with ovalbumin. Airway responsiveness, eosinophils in bronchoalveolar lavage fluid, and lung lymphocyte subsets (CD4 and CD8) were measured 24 hours and 8 weeks after challenge. In further experiments, we treated IFN-KO mice with recombinant IFN- starting 4 weeks after the challenge for 1 week or 4 weeks. Airway responsiveness, bronchoalveolar lavage eosinophils, and lung CD4 cells were increased 8 weeks after challenge in IFN-KO but not WT mice. IFN- treatment returned lung CD4 cell numbers to values obtained in unchallenged mice. One week of IFN- treatment also returned airway responsiveness to baseline levels; however, 4-week treatment with IFN- failed to decrease airway responsiveness below levels observed in untreated animals. This suggests that IFN- plays an essential role in reversing allergen-induced airway inflammation and hyperresponsiveness and that it may have dual actions on the latter. Observations that IFN- reverses airway responses, even when administered after challenge, suggests that IFN- treatment could control allergic disease, including asthma. Keywords: asthma; bronchial hyperreactivity; airway inflammation

In spite of increasing evidence suggesting its suppressive action against allergic responses, the role of IFN- in these responses is still controversial and unknown. For example, allergen-induced response in IFN-deficient (IFN-KO) and IFN- receptor-deficient mice have yielded conflicting results, leading to conclusions that IFN- either prevents or prolongs allergic airway responses, respectively (15, 16). Thus, in the present study we first measured and compared markers of airway inflammation and function, lung T lymphocyte subsets, bronchoalveolar lavage (BAL) eosinophilia, and airway responsiveness to methacholine (MCh) in IFN-KO and wild-type (WT) mice at several time points after airway allergen challenge. Results indicate that both airway inflammation and airway hyperresponsiveness showed persistent increase in IFN-KO animals. In the second series of experiments, we observed whether or not exogenous IFN- administered after allergen challenge can reverse the prolonged allergic airway responses in sensitized and challenged IFN-KO mice.

METHODS
Animals
Female BALB/c IFN-KOdeficient and WT mice (810 weeks of age; Jackson Laboratories, Bar Harbor, ME) were housed in specific pathogenfree conditions for 1 week before experimental use. All procedures were approved by the Animal Research Ethics Board at McMaster University.

Chronic inflammation, including eosinophilia, is thought to play a major role in the pathogenesis of bronchial asthma. Recently it has become widely accepted that T helper (Th) lymphocytes orchestrate this disorder through producing specific cytokines. Among these cytokines, those from Th2 lymphocytes, such as interleukin (IL)-4, IL-5 and IL-13, have a pivotal role on development of allergic inflammation in asthma (1, 2). There is accumulating evidence that Th1 cytokines, such as interferon- (IFN-) and IL-12, suppress and counteract this Th2 response and vice versa (36). Thus an imbalance between Th1 and Th2 cytokines toward Th2 predominance is thought to underlie various asthmatic reactions. The suppressive effects of IFN- have been shown to be mediated by various mechanisms, such as inhibiting Th2 cytokine production and skewing the differentiation of naive T cells toward Th1 subtype preference (4, 710). In animal models, treatment with IFN- suppresses airway inflammation induced by subsequent allergen challenge in mice (11, 12). Furthermore, recent studies suggest that IFN- might prevent persistence of airway inflammation induced by acute allergen challenge (1114).
(Received in original form February 8, 2002; accepted in final form May 22, 2002) Supported by the Ontario Thoracic Society and the Canadian Institutes for Health Research and the St. Joseph Healthcare Foundation. R.L. is a Canadian Institutes for Health Research Fellow, P.O. is a Canadian Institutes for Health Research Senior Scientist, and M.I. is the Harbinger Scholar in Respiratory Medicine. Correspondence and requests for reprints should be addressed to Dr. Mark D. Inman, Firestone Institute, St. Josephs Hospital, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6 Canada. E-mail: inmanma@mcmaster.ca This article has an online data supplement, which is accessible from this issues table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 166. pp 451456, 2002 DOI: 10.1164/rccm.200202-095OC Internet address: www.atsjournals.org

Study Design
To compare the duration of allergic airway responses between IFN-KO and WT mice, both were sensitized and challenged with ovalbumin (OVA) or saline as described previously (17). Outcome measurements were made 24 hours (Day 21) and 8 weeks (Day 76) after OVA challenge (Figure 1). The second series of experiments was designed to determine the effects of treatment with IFN- on the duration of allergic responses in IFN-KO mice. Treatment with either recombinant IFN- (6,000 units/ day, subcutaneous) or diluent (saline) was started 4 weeks after the last challenge (Figure 1). In separate groups, treatment was continued for only 1 week to determine whether ongoing allergic responses could be suppressed by a brief period of treatment. In both cases, outcome measurements were made 4 weeks after the initiation of treatment. Due to laboratory relocation, the first set of experiments was performed at McMaster University, whereas the second set of experiments with IFN- treatment was performed at St. Josephs Health Care.

Airway Responsiveness
Airway responsiveness was measured as in previous work by the authors (17), based on the response of total respiratory system resistance to increasing intravenous doses of MCh, using the flow interrupter technique modified for mice (18). Airway responsiveness was quantified by the slope of the linear regression between peak respiratory system resistance and the log10 of the MCh dose, using the data from the 10, 33, and 100 g/kg doses only.

BAL
BAL fluid (BALF) was collected, processed, and stained as reported previously by the present workers (17). Cell differential counts were performed based on morphological criteria by one investigator, blind to the experimental condition. Enzyme-linked immunosorbent assay analysis for IL-5 and IL-13 was performed on BAL supernatant using

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Figure 1. Study design for sensitization/challenge to OVA or saline, recombinant IFN- treatment and outcome measurements.

commercial kits (BD Biosciences, Oakville, ON, Canada) as described previously (3).

Lung Histology
After BAL, lungs were removed from mice for histologic evaluation of eosinophilia. Fixation and staining techniques were the same as previously reported by the current authors (17).

Digested Lung Cell Collection

After the BALF collection, lungs were removed, minced, incubated for 60 minutes at 37C with Dulbeccos phosphate-buffered saline (PBS), containing 0.4 mg/ml collagenase (type 1A), 330 U/ml hyaluronidase, 50 U/ml DNase, 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin, and then filtered through 52-nm filters. Erythrocytes were removed by lysis with ice-cold NH4Cl-Tris buffer. Cell suspension was adjusted to 1 106 cells/ml with Dulbeccos PBS.

Flow Cytometry
Flow cytometry was performed based on the protocol described by Hogan and colleagues (19). A total of 1 106 cells were incubated with flourescein isothiocyanateanti-mouse CD4 mAbs, Cy-Chrome anti-mouse CD8a mAbs and Phycoerythrinanti-mouse CD90.2 mAbs for 30 minutes and purified anti-mouse CD16/CD32 mAbs underlaid. Analysis was performed on a Becton Dickinson (Franklin Lakes, NJ) FACScan flow cytometer, using Cellquest and WinMDi software packages (BD Biosciences, Oakville, ON, Canada). CD90.2CD4 (CD4), and CD90.2CD8a (CD8) stained cells were identified by detection of FL-1 (flourescein isothiocyanate), FL-2 (Phycoerythrin) and FL-3 (CyChrome) and expressed as percentages of T cells (all antibodies: Pharmingen, Mississauga, ON). Numbers of positive cells were calculated by multiplying total cell count obtained after lung digestion.

Airway responsiveness to MCh was significantly increased in both IFN-KO and WT mice 24 hours after OVA challenge compared with saline challenge in corresponding animal groups (Figure 2A). Furthermore, airway hyperresponsiveness was sustained in IFN-KO mice for at least 8 weeks after OVA challenge without any significant decline compared with 24 hours after the challenge. On the other hand, airway responsiveness returned to baseline values in WT mice 8 weeks after OVA challenge. At this time point, there was a significant difference in airway responsiveness to MCh between IFN-KO and WT mice (Figure 2B). Airway eosinophilia. After saline challenge of both WT and IFN-KO mice, no eosinophils were observed in BALF at either time point investigated (24 hours and 8 weeks after the challenge). Within 24 hours after OVA challenge, prominent eosinophilia was observed in both WT and IFN-KO mice, and IFN-KO mice had almost twofold greater eosinophilia compared with that in WT mice. BAL eosinophilia persisted for at least 8 weeks after OVA challenge in IFN-KO mice, although the number of eosinophils significantly decreased compared with the acute phase of the reaction measured 24 hours after the challenge. On the other hand, eosinophils had completely disappeared from BALF from WT mice at this time point (Figure 3A). Lymphocytes in lungs. In saline-challenged mice, there was no significant difference between WT and IFN-KO mice in numbers of lung CD4 and CD8 T lymphocytes 24 hours after the challenge, although a significant increase was observed in IFN-KO mice 8 weeks after challenge (Figures 3B and 3C). Increased numbers of lung CD4 and CD8 T lymphocytes were detected from both WT and IFN-KO mice 24 hours after OVA challenge compared with saline-challenged animals. Eight weeks after OVA challenge, there was a persistent increase in CD4 lymphocytes in OVA-challenged IFN-KO mice above the level in saline-challenged animals, whereas the increase of CD4 cells had completely reversed in WT mice (Figure 3B). Although change in CD8 T lymphocytes showed a similar trend 8 weeks after OVA challenge, the difference between WT and IFN-KO mice was not significant at that sampling time (Figure 3C).
Effect of Treatment with Recombinant IFN- Protein in IFN-KO Mice

Analysis
Comparisons among each group of mice with respect to airway reactivity (slope of the respiratory system resistance MCh [log transformed] doseresponse curve), BAL eosinophils, and lung T lymphocyte subsets were made using analysis of variance. All post hoc comparisons were performed using Newman-Keuls test for significant effects. All comparisons were two-tailed, with critical set at 0.05.

RESULTS
Allergic Responses in IFN-KO and WT Mice

Airway responsiveness. There were no differences in baseline airway responsiveness to MCh measured 24 hours after saline challenge between IFN-KO and WT mice.

Airway responsiveness. Treatment with recombinant IFN- did not change airway responsiveness in saline-challenged mice compared with diluent treated group. In OVA-challenged mice, data collected 3 weeks after treatment indicated that the 1 week treatment with recombinant IFN- completely reversed airway hyperresponsiveness back to the baseline value. However, paradoxically, longer treatment (4 weeks) with recombinant IFN- had no reversing effects on airway hyperresponsiveness induced by OVA challenge in IFN-KO mice when they were studied 24 hours after the final treatment (Figure 4). Furthermore, 4-week treatment with IFN- tended to increase airway responsiveness compared with diluenttreated controls, although this result was not statistically significant (Figure 4). Airway eosinophilia. In the second series of experiments, no eosinophils were found in any BALF samples or in H&Estained airway sections. This was true for IFN-KO mice treated with either recombinant IFN- or diluent after challenge either with OVA or saline (data not shown.). Lymphocytes in lungs. In saline-challenged IFN-KO mice, treatment with recombinant IFN- for 4 weeks had no effect on numbers of lung CD4 T cells compared with diluent-treated animals.

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Figure 2. Airway responsiveness to MCh in IFN-KO and WT mice sensitized and challenged with OVA or saline. (A) Doseresponse relationship of total respiratory system resistance (RRS) to increasing intravenous doses of MCh. (B) From the RRSMCh doseresponse curve in A, an index of airway responsiveness was calculated as the slope of the linear regression between peak RRS and the log10 of the MCh dose, using the data from the 10, 33, and 100 g/kg doses only. Each group consists of 810 mice. *p 0.05 compared with saline-challenged animals. ! p 0.05 compared with WT animals.

As with the first series of experiments, there was a prominent and significant increase in CD4 T cells 8 weeks after OVA challenge in IFN-KO mice compared with saline-challenged mice. Treatment with recombinant IFN-, both for 1 week and 4 weeks, completely reversed the increased numbers of lung CD4 T lymphocytes to the baseline levels observed in saline-challenged mice. There was no significant difference between the two treated groups (Figure 5A). Allergen challenge and subsequent treatment with recombinant IFN- had the same effect on CD8 T lymphocyte numbers as on CD4 T cells (Figure 5B). IL-5 and IL-13 concentration in BALF. To investigate the mechanisms underlying the above observations, we measured concentration of cytokines: IL-5 and IL-13 in BALF from IFNKO mice after allergen or saline challenge and treatment either with recombinant IFN- or diluent. In diluent-treated mice there was a significant increase in IL-5 after OVA challenge compared with saline challenge. In OVA-challenged mice, treatment with both 1 week and 4 weeks of IFN- reversed this increase in IL-5 in OVA-challenged mice, whereas recombinant IFN- protein had no significant effects on IL-5 levels in salinechallenged animals (Figure 5C). IL-13 levels were not different between any challenge or treatment groups (data not shown).

Figure 3. Eosinophils in BALF and whole lung T lymphocyte subsets in IFN-KO and WT mice sensitized and challenged with OVA or saline. Positive cells were expressed as percentages of T cells, and numbers of positive cells were calculated by multiplying total cell count obtained after lung digestion. (A) Eosinophils in BALF. (B) Thy1.2CD4 (CD4) cells. (C) Thy1.2CD8 (CD8) cells. Each group consists of 810 mice (A) and 5 mice (B, C).*p 0.05 compared with saline challenged animals. !p 0.05 compared with WT animals. #p 0.05 compared with 24 hours after the challenge.

DISCUSSION
In the present study, we observed persistent hyperresponsiveness and inflammation of airways in sensitized IFN-KO mice

after allergen challenge, whereas these responses were transient in WT animals. In addition, we observed that depending on the treatment protocol, exogenous IFN- protein could reverse these ongoing reactions. To our knowledge, this is the first report to reveal the direct suppressing effect of IFN- against an ongoing asthmatic-type reaction. Although the treatment with IFN- constantly reversed allergic airway inflammation, the physiologic consequence of this treatment differed depending on treatment duration and proximity to the measurement of airway responsiveness. These observations suggest a possible pleiotropic mechanism underlying the action of this cytokine. Our observation that BAL eosinophils, lung CD4 cells, and airway hyperresponsiveness (AHR) were persistent 8 weeks after challenge only in IFN-KO mice strongly suggests that this cytokine may play an important role in preventing persistence of both airway inflammation and hyperresponsiveness after a brief period of exposure to a sensitized antigen. This observation is in agreement with previous findings in IFN-receptor KO mice (15), but is completely opposite to previous findings in IFN-KO mice by Hofstra and colleagues (16). In that study, allergic responses were suppressed in the IFN-KO mice. The reason for the different findings between

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Figure 4. Airway responsiveness to MCh in sensitized IFN-KO mice after treatment with either recombinant IFN- protein (1 week or 4 weeks) or diluent (4 weeks). Upper panel: Doseresponse relationship of total respiratory system resistance (RRS) to increasing intravenous doses of MCh. Lower panel: From the RRSMCh doseresponse curve in upper panel, an index of airway responsiveness was calculated as the slope of the linear regression between peak RRS and the log10 of the MCh dose, using the data from the 10, 33, and 100 g/kg doses only. Each group consists of 810 mice. *p 0.05 compared with salinechallenged animals. !p 0.05 compared with diluent-treated animals.

Figure 5. Whole lung T lymphocyte subsets and IL-5 concentration in BALF in sensitized IFN-KO mice after treatment with either recombinant IFN- protein (1 week or 4 weeks) or diluent (4 weeks). Positive cells were expressed as percentages of T cells, and numbers of positive cells were calculated by multiplying total cell count obtained after lung digestion. (A) Thy1.2CD4 (CD4) cells in lung; (B) Thy1.2CD8 (CD8) cells in lung; (C) IL-5 concentration in BALF. Each group consists of five mice. *p 0.05 compared with saline-challenged animals. ! p 0.05 compared with diluent-treated animals.

these laboratories is unclear, but consistent with our findings there is considerable evidence supporting mechanisms whereby exogenous IFN- may play a regulatory role in the extent of airway responses to allergen. Some of the mechanisms that may be relevant include suppressing the release of Th2-type cytokines from activated T cells (4, 10), suppressing differentiation of naive T cells to Th2 subtypes (79), facilitating apoptosis of T cells and eosinophils (2022), suppressing local recruitment of eosinophils (23, 24), and inducing nitric oxide production, which might suppress contraction of airway smooth muscle (25). Whether the lack of any of these responses is enough to account for the persistence of ongoing inflammation or AHR in the IFN-KO mice is not clear. Clearly other possibilities exist. For example, the Th2-type response to OVA may have resulted in the mice becoming sensitized to other environmental antigens. However, exploring the specific nature of the ongoing response was beyond the scope of our investigations. There was a significant increase in lung CD4 cells in the saline-challenged IFN-KO mice studied at 8 weeks compared with those studied 24 hours after challenge. The relevance of this observation is not clear; there was no increase in

BAL cellular inflammation, nor was AHR observed in these mice. Whether this observation reflects a unique phenomenon in the aging of the IFN-KO mouse needs to be confirmed. A surprising observation in the second series of experiments was that we no longer observed a sustained eosinophilia in BALF or lung tissue of IFN-KO mice after allergen challenge, despite the fact that persistent AHR and increases in IL-5, CD4 and CD8 cells. Although we cannot be sure of the mechanisms of this effect, it appears likely that the stress of daily injections may have interfered with some aspects of mechanisms underlying the ongoing eosinophilia. Interestingly, daily injections with saline did not reverse other aspects of the sustained response to allergen in the IFN-KO mice, including airway hyperresponsiveness, BALF IL-5 levels, and elevations in lung CD4 and CD8 cells. In this model at least, it would appear that resolution of eosinophilia might occur despite an ongoing physiologically relevant Th2-type response. Discrepancy between the ongoing BAL IL-5 responses and suppression of BAL eosinophilia suggests that the mechanisms responsible for suppression of eosinophilia did completely suppress IL-5 production, although it must be pointed out that levels of IL-5 observed in the saline-treated mice 8 weeks after allergen challenge were much lower than normally observed in mice directly after allergen challenge (3).

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The major novel finding in the present study is that 1 week of treatment with exogenous recombinant IFN- reversed the persisting airway responses to allergen, including BALF IL-5 levels, lung CD4 and CD8 cells, and AHR. This was true even though treatment was started 4 weeks after allergen challenge, a time when the allergic airway reaction was clearly established. There have been several reports on the effect of IFN- treatment initiated before sensitization or allergen challenge on preventing allergic reactions in mice (1114). Additionally, Huang and coworkers showed a reversing effect of OVA-specific Th1 cells transferred from sensitized rats after allergen challenge and further demonstrated that this effect was IFN-dependent (26). Although results of the present study are consistent with these observations, to our knowledge, ours is the first illustration that treatment with IFN- may be useful in reversing asthmatic-type reactions that are already established. This observation is consistent with the concept that the duration of a response to allergen is to some extent regulated by IFN-, and also greatly supports the potential use of IFN- in the management of Th2-type diseases, including allergy and asthma. Although IFN- treatment may prove to be useful in the management of several inflammatory conditions, our observation that the reversal of AHR was dependant on the timing of the treatment indicates that more needs to be learned about the multiple effects of this cytokine before a clear role in the management of the patient with asthma can be established. Whereas 4 weeks of treatment with IFN- was just as effective at reversing markers of an ongoing inflammatory response to allergen (BAL IL-5 and lung CD4 and CD8 cells), AHR was still present in these mice at the time they were killed. The observation that AHR is independent of cellular markers of inflammation in allergen-challenged mice is not new. The observation that the effects of IFN- on airway hyperresponsiveness may vary depending on the timing of treatment is new and deserves further attention. The different manners of changes in AHR and inflammation in the present study are consistent with evidence that, although airway hyperresponsiveness and inflammation are strongly related to each other, these two are regulated at least in part via different mechanisms. Proliferation and recruitment of eosinophils are mainly facilitated by IL-5 (27, 28), whereas IL-13 (29, 30) and possibly other T cell mediators (2) play a pivotal role in the development of airway hyperresponsiveness. It appears to be paradoxical that AHR was resolved in allergen-challenged IFN-KO mice having had a 3-week treatment-free period but was still present in those mice currently receiving IFN- treatment. Clearly, this observation is not explained by a simple induction of AHR by ongoing IFN- treatment, as mice that had not been exposed to allergen but were receiving IFN- treatment had normally responsive airways. Thus, this finding indicates that the timing or duration of treatment with IFN- of allergen-challenged mice had a major influence on whether or not there was reversal of the allergen-induced AHR. Although the reason for the difference in treatment effects is unclear, a plausible explanation is that treatment of allergen-challenged mice with IFN- resulted in a repolarization of the ongoing Th2-type response, possibly toward more of a Th1 direction. Thus, the mechanism of AHR observed in the mouse studies immediately after 4 weeks of treatment may have been based on a Th1-type response, as has been observed previously (31, 32). In the mice that were observed 3 weeks after 1 week of IFN- treatment, this treatment-free period may have allowed resolution of this repolarized response. Clearly, at this stage this is a hypothesis and will need to be addressed in further experiments. Whatever the

underlying mechanism, these results indicate that the effects of IFN- treatment of allergic disease may be complex, and that depending on the timing and dosing of treatment, suppression of typical Th2 responses may or may not be associated with functional improvement. Although we have not been able to clearly describe the mechanisms of sustained AHR in the allergen-challenged IFN-KO mice, several possibilities exist. One likely explanation is that ongoing Th2-type responses result in the production of mediators known to play a role in immune mediated AHR. The most likely candidate, IL-13, was not detectable in these mice at the time of the sustained AHR. Use of the antiCD4 antibody, as has been done in WT mice (19), may provide further insight into whether the sustained AHR was mediated through an ongoing immune response. A second possibility is that the prolonged inflammatory response in the IFNKO mice resulted in a functionally important remodeling of the airway wall. Although we have not assessed structural changes in the airway of these mice, we would argue that this is unlikely the cause of the sustained AHR, given that 1 week of IFN- protein treatment was able to completely reverse airway dysfunction. In conclusion, the results of the present study clearly revealed a crucial role of IFN- to reverse ongoing allergic reactions in a mouse model. Thus, clinical use of this cytokine may be effective in managing ongoing allergic conditions such as bronchial asthma. Nevertheless, apparently pleiotropic actions of IFN-, with variable effects on AHR, indicates that further investigation is necessary before establishing or recommending its clinical usage on allergic diseases.
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