Splice isoforms of human interleukin-4 are functionally active in mice in vivo

Abstract

Interleukin-4 (IL-4) acts on cultured cells in a species-specific fashion, although several reports have suggested that human (h) IL-4 may be functionally active in rodents in vivo. The latter finding, if true, would not only offer possibilities for pre-clinical testing of novel hIL-4-targeting therapies in animals, but also suggests new opportunities for mechanistic studies of IL-4 and its receptors. Conventional IL-4 is encoded by four exons, whereas its poorly studied alternatively spliced isoform is encoded by exons 1, 3 and 4 (IL-4δ2). Replication-deficient adenovirus-mediated gene delivery of hIL-4 isoforms (hIL-4 or hIL-4δ2) to mouse lungs caused similar pulmonary infiltration of T and B lymphocytes, but not eosinophils. There were significant differences in the changes of pulmonary cytokine milieu induced by hIL-4 compared with hIL-4δ2, with hIL-4δ2 inducing higher levels of pro-inflammatory (tumour necrosis factor-α, IL-1, and monocyte chemotactic protein-1) and T helper type 1 (IL-12 and interferon-γ) cytokines. There was no elevation in endogenous mouse (m) IL-4 or mIL-4δ2 mRNAs, and germ-line deficiency of mIL-4 did not affect the degree of pulmonary infiltration. When combined with an ovalbumin model of asthma, hIL-4δ2 stimulated a greater accumulation of lymphocytes than did hIL-4. Pulmonary infiltration of lymphocytes induced by expression of hIL-4 or hIL-4δ2 was attenuated, but not completely abrogated, by germ-line deficiency of mIL-4Rα or murine signal transducer and activator of transcription 6, suggesting that these signalling molecules mediate the in vivo effects of hIL-4 isoforms in mice. These findings suggest that splice isoforms of human IL-4 are functionally active in vivo in mice, and partially share the effects of the corresponding species-specific isoforms.

Introduction

Interleukin-4 (IL-4) is a pleiotropic cytokine and a multifunctional regulator of the immune system; it also controls growth, survival and gene expression in other cell types.¹ Interleukin-4 regulates differentiation of antigen-stimulated naive T helper cells towards a type 2 phenotype (Th2), and is produced by activated Th2 cells, mast cells, basophils and eosinophils. It induces proliferation of T lymphocytes, inhibition of apoptosis and immunoglobulin class switching in B cells, alternative activation of macrophages, and production of collagen by fibroblasts. Diverse disease processes, including allergic disorders, asthma, parasitic infections, tuberculosis, pulmonary fibrosis and systemic connective tissue diseases, are thought to involve IL-4 as an important pathogenetic player.^2–6 These observations suggest that IL-4 may be a viable therapeutic target in numerous diseases. However, progress in this direction has been limited by at least two obstacles. First, functional activity of human (h) IL-4 appears to be species-specific in cell culture, preventing easy testing of anti-hIL-4 therapies in mice. Second, this cytokine naturally occurs in two splice isoforms, one of which, the conventional IL-4, is encoded by four exons and has been well studied, whereas the other isoform, omitting exon 2 and encoded by exons 1, 3 and 4, has not been thoroughly investigated.

Although hIL-4 appears to have no effect on the proliferation of cultured mouse or rat T lymphocytes,^7,8 structural analysis suggests that the two key residues of hIL-4 that define its binding to hIL-4Rα, namely Glu-9 and Arg-88, as well as the minor determinants, Arg-53 and Arg-85, are all present in mouse (m) IL-4.⁹ Direct experiments in the BIAcore system have revealed that the normally glycosylated mIL-4 directly binds to hIL-4 receptor α (Rα).⁹ Moreover, several previous reports have shown that hIL-4 is active in mice or rats in vivo in a fashion similar to that of mIL-4.^10–12 It is therefore possible that hIL-4 may be at least partially active in vivo in mice.

A naturally occurring alternatively spliced variant of IL-4, termed IL-4δ2 (IL-4δ2), was identified in the 1990s,^13,14 and has subsequently been shown to occur naturally in humans, other primates, and various other species, including mice.^15–17 Interleukin-4δ2 lacks the region encoded by the second exon for IL-4, and the corresponding part of the protein comprising 16 amino acids is absent. The in vivo role of IL-4δ2 has not been studied in an animal model, although involvement of IL-4δ2 in disease processes in humans has been suggested by numerous studies. We have reported that expression levels of IL-4δ2 (absolute and relative to IL-4) are increased in pulmonary T lymphocytes in patients with scleroderma lung disease,⁶ and others reported a similar increase in the peripheral blood mononuclear cells of scleroderma patients.¹⁸ In patients with pulmonary tuberculosis, greater absolute expression levels of both IL-4 and IL-4δ2 in blood and lung were associated with more extensive disease.^19–21 Expression of IL-4δ2 was increased in respiratory tract tissues of patients with asthma,²² and pulmonary fibroblasts have been reported to alter the IL-4δ2 : IL-4 ratio in mast cells in patients with asthma.²³ A relative increase in IL-4 and a decrease in IL-4δ2 have been associated with better survival in patients with severe sepsis.²⁴

In this study, we used a replication-deficient adenovirus system^25–27 for gene delivery to mouse lung to over-express human IL-4 or IL-4δ2 in vivo. We show that hIL-4 and hIL-4δ2 partially share functional activities in mice in vivo with their species-specific counterparts, but they differ from each other in that over-expression of hIL-4δ2 appears to have a more pronounced pro-inflammatory effect. These results form the basis for at least limited testing of anti-human IL-4 therapies in mice, as well as creating new opportunities for further basic studies on the biology of IL-4 and IL-4δ2.

Materials and methods

Adenoviral constructs

Adenoviral constructs encoding hIL-4, hIL-4δ2, mIL-4 or mIL-4δ2 were created and validated identically to the previously reported constructs.^25–27 Briefly, GenBank consensus sequences for these cytokines were used to artificially synthesize (GenScript, Piscataway, NJ) the DNA fragments corresponding to the respective cDNAs. The fragments were subcloned into a shuttle vector, and transferred into a recombinant replication-deficient adenovirus vector (AdV) using RAPAD® technology (ViraQuest, North Liberty, IA). The constructs were composed to ensure expression of the cytokines under control of the cytomegalovirus promoter. The validity of the constructs was confirmed by direct automated sequencing. The adenoviruses were purified over two rounds of CsCl gradients. The resultant purified virus particles had a concentration of approximately 1 × 10¹² particles/ml and an infectious titre of 4 × 10¹⁰ plaque-forming units/ml. All viruses, including control AdV-NULL that did not encode a cytokine, encoded green fluorescent protein (GFP) in their backbone under the control of a separate promoter. Infectivity of the viruses was confirmed by GFP fluorescence of infected HEK293 cells in culture, and transcription was confirmed by real-time quantitative PCR with primers specific for the respective mRNAs. Pre-validated primers for all human and mouse IL-4 splice isoforms were purchased from SABiosciences (Frederick, MD), and the primer specificity was further validated by direct sequencing of PCR products. Separately, cell culture supernatants were analysed for production of recombinant cytokines using three independent techniques: (i) Western blotting assays were performed using previously identified monoclonal antibodies that detect both hIL-4 and hIL-4δ2 (indiscriminate antibody) or only hIL-4 (selective antibody), (ii) sandwich ELISA assays using the same antibodies were performed to evaluate the concentration of the secreted recombinant cytokines, and (iii) cell culture supernatants were concentrated, electrophoretically separated, and the fraction at molecular weights (MW) 10 000–20 000 was excised and analysed by liquid chromatography/mass spectrometry assays, confirming production and secretion of IL-4δ2 or IL-4 proteins.

Animals and treatments

Wild-type and germ-line-deficient female C57BL/6 mice aged 10–12 weeks (The Jackson Laboratory, Bar Harbor, ME) were used in this study. The animals were treated in accordance with a research protocol that has been approved by the Institutional Animal Care and Use Committee of the University of Maryland. The adenoviruses were instilled intra-tracheally as previously described in detail.^25–27 Fluorescence microscopy of unstained lung sections for GFP was used to confirm infectivity in vivo. Quantitative PCR of lung homogenates was used to confirm over-expression of mRNAs for the delivered cytokines. The ELISA of pulmonary homogenates or BAL samples from these mice were used to confirm the over-expression of the delivered cytokines at days 7, 14 and 21. Bronchoalveolar lavage (BAL), pulmonary homogenates, histological and immunohistological analyses, and flow cytometry were all performed as previously described.^25–27 Multiplex analyses (Luminex, Austin, TX) of cytokine levels in BAL and lung homogenates were performed in triplicate in each sample from each animal, and the mean values and standard deviations were calculated for the triplicates.

Ovalbumin model of asthma

The ovalbumin (OVA) model of asthma was developed as previously described.^28,29 Briefly, wild-type mice were immunized with aluminium hydroxide alone or 100 μg OVA/alumimium hydroxide by intraperitoneal injection on day 1 and boosted with the same reagents on day 6. Mice were then exposed to 1% OVA in PBS by nebulization for 20 min each day on days 12 and 14. Finally, mice were killed on day 16, and analyses of BAL differential cell counts and of histological changes were performed. This standard protocol was modified by an additional step on day 8, at which point mice were intra-tracheally challenged with recombinant AdV or PBS.

Statistical analyses

Data were processed using Statistica software (StatSoft, Tulsa, OK). Groups were compared using Student's two-tailed t-test, the Mann–Whitney U-test, or analysis of variance (anova), as indicated in the text. For all analyses, P values ≤ 0·05 were considered significant.

Results

Replication-deficient adenovirus-mediated gene delivery

All replication-deficient AdV constructs (AdV-hIL4, AdV-hIL4δ2, AdV-mIL-4, AdV-mIL4δ2, and AdV-NULL) also encoded GFP under the control of a separate promoter and caused infection in cultured HEK 293 cells and in cultured human, mink and mouse lung epithelial cells. Infectivity of the viruses, including AdV-NULL, was evident from GFP fluorescence of the infected cells at 24–72 hr after the virus had been added to the cultures, similar to a previous report (see figure 1a in ref. 25). Delivery of the encoded genes was confirmed by RT-PCR with specific primers. Cells infected with AdV constructs encoding hIL-4 or hIL-4δ2 secreted these cytokines into the supernatants as revealed by Western blotting assays, whereas no IL-4 or IL-4δ2 was detected in AdV-NULL-infected cells (Fig. 1a). Production of hIL-4 or hIL-4δ2 proteins by these cells was also confirmed by liquid chromatography/mass spectrometry of the cell culture supernatant electrophoretic fraction at 10 000–20 000 MW.

Figure 1.

Replication-deficient adenovirus-mediated delivery of the alternatively spliced variant of interleukin-4 (IL-4δ2) and IL-4 in cell culture and in vivo. (a) Western blotting assays of cell culture supernates of HEK293 cells infected with adenovirus vector (AdV) encoding human (h) IL-4, hIL-4δ2, or not encoding a cytokine (NULL). In all cases, supernatants were collected from equal numbers of cultured cells infected with equal viral plaque-forming units (PFUs); cells in all cultures were > 95% infected, based on green fluorescent protein (GFP) fluorescence. In the upper gel, a commercial selective anti-hIL-4 monoclonal antibody detects hIL-4, but not hIL-4δ2. The upper band at approximately 16 000 MW corresponds to the naturally glycosylated form of IL-4, whereas the lower band at approximately 12 000 MW corresponds to the non-glycosylated form. The commercial recombinant human IL-4 (cIL-4) positive control is of bacterial origin and therefore is not glycosylated. In the lower gel, a commercial indiscriminate monoclonal antibody detects both hIL-4 and hIL-4δ2. Enzymatic deglycosylation of hIL-4δ2 (rightmost band) shifts the band from approximately 16 000 MW to the predicted molecular weight of approximately 12 000 MW (based on amino acid composition), further confirming the identity of this protein. (b) ELISA of lung homogenates for indicated proteins on day 7 after intra-tracheal infections with recombinant adenoviruses encoding corresponding proteins. In AdV-hIL-4δ2- and AdV-hIL-4-infected mice, an indiscriminate antibody detecting both hIL-4δ2 and hIL-4 was used. In the AdV-NULL- and AdV-mIL-4-infected mice, ELISA for mIL-4 (R&D Systems, Minneapolis, MN) is shown.

Intra-tracheal instillation of the adenoviral constructs in vivo resulted in pulmonary expression of GFP, as confirmed by Western blotting. The RT-PCR analyses of lung tissue homogenates using specific primers revealed delivery of desired mRNAs for hIL4, hIL4δ2, mIL4, or mIL4δ2. ELISA of pulmonary homogenates or BAL samples from these mice confirmed high levels of protein expression of hIL-4, hIL-4δ2 and mIL-4, whereas no IL-4δ2 or IL-4 protein was detected in the AdV-NULL-infected mice (Fig. 1b). As no antibody against mIL-4δ2 has yet been identified, delivery of mIL-4δ2 protein was deduced based on the results of RT-PCR analyses and on the design of the AdV-mIL4δ2 construct that was similar to other constructs in this study. The kinetics of IL-4 or IL-4δ2 expression was similar to that of a different cytokine in our previous report on adenovirus-mediated gene delivery (see figure 1 in ref. 25). There was no IL-4 or IL-4δ2 protein expression on day 3; high levels of expression were observed on day 7 (Fig. 1b); protein expression declined on days 14 and 21 after intra-tracheal instillations, with trace amounts detectable on day 28. These observations suggest that the stability of the delivered proteins is probably limited to several days and that the model allows for efficient, yet relatively short-term, gene delivery.

Gene delivery of hIL-4 or hIL-4δ2 to mouse lung induces changes in bronchoalveolar lavage cell content

Total and differential BAL cell counts were assessed in mice over-expressing hIL-4 or hIL-4δ2. The total BAL cell counts did not differ between mice infected with AdV-NULL or those infected with the encoding viruses at 7 or 14 days (P > 0·05 by one-way anova). However, the relative contributions of macrophages, lymphocytes and eosinophils significantly changed (Fig. 2a). Of note, only mIL-4 caused significant influx of eosinophils in the lungs of mice, whereas hIL-4, hIL-4δ2 and mIL-4δ2 recruited lymphocytes (Fig. 2a). Flow cytometric assays were performed on BAL cells from hIL-4- or hIL-4δ2-over-expressing mice. The BAL lymphocytes were both T cells (CD4⁺ or CD8⁺) and B cells (CD19⁺); there was no significant difference between the IL-4δ2-over-expressing and IL-4-over-expressing mice in the relative proportions of these lymphocytic subsets. The results of BAL flow cytometry for hIL-4-or hIL-4δ2-over-expressing mice are shown in Fig. 2(b). Additional flow cytometry analyses were performed with BAL cells co-stained with antibodies against CD4, CD25 and FoxP3. Among T cells, the contribution of regulatory T cells (CD4⁺ CD25⁺ FoxP3⁺) doubled in hIL-4δ2- and in hIL-4-over-expressing mice compared with AdV-NULL-infected mice (Fig. 2c). In contrast to over-expression of hIL-4δ2 or hIL-4, over-expression of an unrelated protein hCCL18, a potent and highly selective chemoattractant of T lymphocytes,^25–27 did not change the relative levels of regulatory T cells (Fig. 2c).

Figure 2.

Changes in cellular composition of bronchoalveolar lavage (BAL) in mice over-expressing human interleukin-4 (hIL-4) or its alternatively spliced variant hIL-4δ2 on day 14 after gene delivery. (a) Cell counts on Giemsa-stained cytospin slides of BAL from mice over-expressing the indicated mouse (m) or human (h) cytokines. Animals were either wild-type (WT) or germ-line-deficient knockout (KO) for signal transducer and activator of transcription 6 (STAT6) or IL-4 receptor α (IL-4Rα). Statistically significant differences (P < 0·01, two-tailed Student's t-test) from adenovirus vector (AdV) -NULL-infected mice are indicated with single and double asterisks. Data were pooled from two independent experiments, to a total of 8–10 mice per group. Average counts of three separate fields for each mouse were performed by two technicians blinded to the identities of the samples. The remaining cells were macrophages, whereas other cell types (neutrophils and bronchial cells) were present in minimal amounts not exceeding 5% of total BAL cells. The significant decline in lymphocytes in germline-deficient mice compared with WT animals over-expressing the corresponding cytokines are indicated with double asterisks. (b) Flow cytometry analyses for CD8, CD4 and CD19 (gates shown in scatterplots, the percentage of gated cells is shown at the bottom of the panel) in BAL cells from mice infected with AdV-hIL4, AdV-hIL4δ2, or AdV-NULL. The large diagonal sub-population are pulmonary macrophages that are naturally strongly autofluorescent in the broad emission spectral range. (c) Percentage of CD25⁺ FoxP3⁺ cells among CD4⁺ BAL cells from mice instilled with recombinant adenoviruses, as indicated (n = 3 in each group). Significant (P < 0·05) differences from the AdV-NULL group are indicated with asterisks. (d) Total lung hydroxyproline, mean μg/ml ± SD, in mice infected with the indicated constructs.

We then sought to determine whether hIL-4 and hIL-4δ2 exert their effects by signalling through IL-4Rα and/or murine signal transducer and activator of transcription 6 (STAT6), which are the known mediators of species-specific IL-4 activities. Adenoviral delivery of hIL-4 or hIL-4δ2 was performed in STAT6 germ-line-deficient or IL-4Rα germ-line-deficient mice. Over-expression of hIL-4δ2 or hIL-4 in mice deficient for IL-4Rα was manifested by a significant attenuation of lymphocytic infiltration in the lungs (Fig. 2a), suggesting that these two factors contributed to, but did not solely mediate, the cross-species effects of human IL-4 isoforms on mouse lung. Germ-line deficiency of mIL-4 had no effect on the BAL cell count, suggesting that endogenous mIL-4 was not involved in mediating the effects of human IL-4 isoforms on mouse lung.

Over-expression of hIL-4 or hIL-4δ2 has a limited effect on collagen production in mouse lung

Total lung hydroxyproline assays (a surrogate measure of collagen) as well as Sircol assays (a direct measure of collagen) were performed as described^25–27 to determine whether delivery of hIL-4 or hIL-4δ2 may increase collagen accumulation in the lungs. Delivery of either of these isoforms caused a modest fold increase in collagen (not exceeding 1·62 ± 0·22), but these differences did not attain statistical significance (P = 0·08). These changes were observed in two independent experiments, five animals in each experimental group of mice (Fig. 2d).

Over-expression of hIL-4 or hIL-4δ2 induces lymphocytic infiltration in mouse lung

Histologically, instillation of AdV-NULL caused minimal, if any, changes in the lungs of mice, but delivery of either hIL-4 or hIL-4δ2 caused lymphocytic infiltration in the perivascular and peribronchial spaces and within the alveolar septa (Fig. 3a–c). The lymphocytic infiltrates appeared as early as day 3 after infection, increased further on days 7–14, and gradually declined thereafter. Immunohistochemically, the infiltrates consisted predominantly of T and B cells (CD3⁺ and B220⁺, respectively) without macrophages in all animal groups over-expressing these cytokines (Fig. 3d,g); the infiltrates stained similarly for CD4, CD8 and CD19 (not shown). The staining for these markers in wild-type mice was similar in hIL-4 and hIL-4δ2 mice. The infiltrates did not contain macrophages, based on staining for F4/80; macrophages appeared only sparsely throughout the lung parenchyma (not shown) in either hIL-4- or hIL-4δ2-expressing mice. Gene delivery of hIL-4 or hIL-4δ2 to the lungs of IL-4Rα^−/− (Fig. 3e,f) or STAT6^−/− (Fig. 3h,i) mice led to the appearance of infiltrates that were significantly smaller and less dense than those in wild-type mice (Fig. 3). These observations suggested again that STAT6 and IL-4Rα contribute to, but do not solely mediate, the cross-species effects of human IL-4 isoforms on the mouse lung. Germ-line deficiency of mIL-4 had no distinctive effect on histological changes, again suggesting that endogenous mIL-4 was not involved in mediating the effects of human IL-4 isoforms on mouse lung.

Figure 3.

Histological changes caused by over-expression of human interleukin-4 (hIL-4) or its alternatively spliced variant hIL-4δ2 in mouse lung on day 14 after adenovirus vector (AdV) instillation, magnification ×200. Mice were wild-type (WT; a–d, g), IL-4 receptor α deficient (IL-4Rα^−/−; e, f), or signal transducer and activator of transcription 6-deficient (STAT6^−/−; h, i). Sections from mice infected with AdV-NULL (a), hIL-4 (b, e, h), or hIL-4δ2 (c, d, f, g, i) were stained with haematoxylin & eosin or immunohistochemically for CD3 (d) or B220 (g). The infiltrates in WT mice are indicated with arrows, whereas substantially smaller and less dense infiltrates in germ-line-deficient mice are indicated with arrowheads. Similar observations were made in at least five mice in each group.

Over-expression of IL-4δ2 affects the pulmonary cytokine milieu

To determine whether hIL-4 or hIL-4δ2 over-expression affects the overall pulmonary cytokine milieu, mice were infected with each of the adenoviral constructs and killed after 14 days to obtain pulmonary homogenates or BAL samples in three independent experiments. The multiplex cytokine assays tested levels of pro- and anti-inflammatory cytokines, as well as Th1 and Th2 cytokines (Table 1). In addition to the data presented in Table 1, no significant differences were found in BAL between mice over-expressing hIL-4δ2, hIL-4 or NULL for IL-1β, IL-12p70, IL-13, eotaxin or IL-1α. Similarly, there was no difference between these groups for transforming growth factor-β in lung homogenates, or IL-5 in either BAL or lung homogenates. Importantly, endogenous mIL-4 or endogenous mIL-4δ2 mRNAs were not detectable in either BAL or lung homogenates of mice over-expressing hIL-4 or hIL-4δ2, further suggesting that the observed effects on mouse lung were not mediated by induction of endogenous mouse IL-4δ2 or IL-4. These observations suggest that gene delivery of human IL-4 splice isoforms elicits changes in the pulmonary cytokine milieu.

Table 1.

Differences in cytokine levels measured by Luminex in lung homogenates (Lung) or bronchoalveolar lavage (BAL) of mice over-expressing human interleukin-4 (hIL-4) or its alternatively spliced variant hIL-4δ2 on day 14 after infection with the indicated adenoviruses

		hIL-4		hIL-4δ2		NULL
Cytokine	Sample	Median pg/ml [1st, 3rd quartile]	n	Median pg/ml [1st, 3rd quartile]	n	Median pg/ml [1st, 3rd quartile]	n
TNF-α	Lung	67 [25, 84]*	13	112 [89, 135]**	13	0 [0, 12]	5
TNF-α	BAL	20 [9, 29]*	14	31 [19, 40]**	14	0 [0, 0]	5
KC (IL-8)	Lung	247 [222, 261]*	3	258 [256, 297]*	3	125 [108, 142]	3
KC (IL-8)	BAL	61 [49, 101]*	6	54 [43, 79]*	6	0 [0, 0]	3
IL-1α	Lung	232 [151, 324]*	12	341 [288, 448]**	13	89 [81, 135]	5
TGF-β	BAL	84 [63, 97]*	14	81 [64, 108]*	14	43 [6, 75]	5
IFN-γ	Lung	0 [0, 43]	13	64 [49, 75]**	13	0 [0, 0]	5
IFN-γ	BAL	0 [0, 9]	14	16 [12, 25]**	14	0 [0, 0]	5
IL-12p40	Lung	68 [63, 68]*	3	131 [88, 158]**	3	13 [7, 20]	3
IL-12p40	BAL	154 [136, 169]*	6	186 [159, 190]**	6	60 [48, 71]	3
MCP-1	Lung	0 [0, 2]	10	8 [1, 17]**	12	0 [0, 0]	9

Single asterisks indicate significant differences (P < 0·05, Mann–Whitney U-test) from the AdV-NULL-infected animals. Double asterisks indicate significant differences from both AdV-NULL-infected animals and from animals infected with the other construct (AdV-IL-4δ2 or AdV-IL-4). Data combined from one to three independent experiments performed by two different technicians, with the total number of animals (n) indicated for each cytokine in each group

TNF-α, tumour necrosis factor-α; TGF-β, transforming growth factor-β; IFN-γ, interferon-γ; MCP-1, monocyte chemoattractant protein 1.

Effects of human IL-4 or IL-4δ2 on the mouse OVA model of asthma

Considering the well-known role of IL-4 in the pathophysiology of asthma, further experiments were performed using the OVA-induced allergic asthma model (see Materials and methods) combined with over-expression of hIL-4 or IL-4δ2. On day 8 after OVA immunization, mice were intra-tracheally challenged with AdV-hIL-4, AdV-hIL-4δ2, AdV-Null, or PBS. Over-expression of hIL-4 or hIL-4δ2 significantly increased the degree of OVA-induced eosinophilia, and furthermore, hIL-4δ2 caused a more pronounced accumulation of lymphocytes and neutrophils than hIL-4 (Fig. 4). These data further suggest that human IL-4 isoforms not only induce lymphocytic inflammation in the lung, but also worsen allergic inflammation.

Figure 4.

Changes in the lungs of ovalbumin (OVA) -sensitized mice additionally challenged with the alternatively spliced variant of human interleukin-4 (hIL-4δ2) or hIL-4 over-expression (day 7 after infection with adenoviral constructs, n = 4 per treatment group). (a) Histologically (haematoxylin & eosin staining, × 100 magnification), over-expression of hIL-4δ2 or hIL-4 worsens pulmonary inflammation compared with AdV-NULL-infected, OVA-sensitized mice. (b) Bronchoalveolar lavage (BAL) differential cell counts show modest, yet statistically significant (asterisks), increases in eosinophils in OVA-sensitized mice over-expressing hIL-4δ2 (P = 0·1) or hIL-4 (P = 0·02) compared with OVA-sensitized AdV-NULL-infected mice. Lymphocytes were similarly increased by either hIL-4δ2 or hIL-4 delivery (P = 0·01 in both cases compared with OVA-sensitized AdV-NULL-infected mice). Delivery of hIL-4δ2 had a more profound effect on accumulation of pulmonary lymphocytes compared with hIL-4 delivery (P = 0·01). The number of neutrophils was also significantly higher in the hIL-4δ2-over-expressing mice compared with mice over-expressing hIL-4 (P < 0·01).

Discussion

In this study, over-expression of human IL-4 or IL-4δ2 in vivo in mice (see Fig. 1) caused changes in pulmonary cellularity, particularly accumulation of T and B lymphocytes (see Figs 2a,b, and 3) and in the cytokine milieu (see Table 1). Both splice variants similarly increased the pulmonary content of CD25⁺ CD4⁺ FoxP3⁺ cells (see Fig. 2c), consistent with the previous observation that signalling through the IL-4 receptor induces generation of such cells extrathymically from CD25⁻ CD4⁺ precursors.³⁰

There was also a tendency for collagen accumulation (see Fig. 2d). Although the significance of differences in total lung collagen did not reach the 0·05 threshold and remained at P = 0·08 in each of the independent experiments or when the data were combined from two separate experiments, this observation suggests that a more profound profibrotic effect might be observed upon a more prolonged expression of hIL-4 or hIL-4δ2 in mouse lung.

Germ-line deficiency of IL-4Rα or STAT6 attenuated the effects of hIL-4 or hIL-4δ2 over-expression in lung (Fig. 3). There was no eosinophilia in response to delivery of either hIL-4 or hIL-4δ2 to mouse lung, suggestive of a partial, but incomplete, cross-species activity between human and mouse IL-4 in vivo. However, over-expression of hIL-4 or hIL-4δ2 worsened the severity of allergic inflammation, particularly accumulation of eosinophils, lymphocytes, and neutrophils in the lung (see Fig. 4).

There were no changes in the levels of endogenous mIL-4 or mIL-4δ2 mRNAs following delivery of hIL-4 or hIL-4δ2, and gene knockout mice deficient in mIL-4 were indistinguishable from wild-type mice in their responsiveness to gene delivery of hIL-4 or hIL-4δ2. These observations suggest that the isoforms of human IL-4 act on mouse lung in vivo directly, without a mediating effect of endogenous IL-4.

These observed effects were probably specific cytokine-induced changes and not adenoviral effects for the following reasons. First, mice instilled with AdV-NULL showed minimal, if any, infiltration of cells in the lungs, suggesting that the response to the infection with the non-replicating adenovirus was minimal within the timeframe of the experiments. Second, all adenoviral constructs encode a foreign protein (GFP) in their backbone under a different promoter, leading to strong GFP expression in cells infected with any of these viruses.²⁵ AdV-NULL-infected mice over-expressing GFP in their lungs²⁵ show minimal if any pulmonary response, suggesting that over-expression of a foreign protein has minimal effect on the lung within the experimental timeframe. Third, delivery to mouse lung of an unrelated protein, chemokine CCL18, using the same adenoviral system, resulted in a highly selective accumulation of T cells, but not B cells, and in minimal disturbance of the pulmonary cytokine milieu.^25–27 In contrast, delivery of hIL-4 or hIL-4δ2 to the lungs resulted in the accumulation of both T and B cells, and in substantial changes in the cytokine milieu. These observations suggest that the pulmonary outcomes are specific to the proteins being delivered and are different from an overall inflammatory response or broad immune activation.

There are, however, limitations to adenovirus-mediated gene delivery, despite its broad use in animal models and the rapid progress toward its use as a routine treatment in the clinical setting.³¹ In this study, the adenoviruses most likely infected pulmonary epithelial cells, and these cells do not produce IL-4 or IL-4δ2 in health or disease. An alternative approach could be a transgenic model of T-cell-specific inducible expression of IL-4δ2, but it would be difficult to ensure the expression of IL-4δ2 in the lung only, because of the systemic presence of T cells and the unavailability of molecular markers distinguishing pulmonary T cells. Another limitation is that we addressed numerous cytokines whose levels changed or did not change in response to IL-4 or IL-4δ2 gene delivery (see Table 1 and related text), but the data do not specifically identify the factors that drive lymphocytic infiltration of the lung, and do not specify whether resident pulmonary cells, infiltrating lymphocytes, or both produce these cytokines. Considering the pleiotropic effects of the cytokines identified in Table 1 and the ability of diverse cell types to produce these cytokines, it is likely that the complexity of the in vivo situation is such that IL-4 or IL-4δ2 drive lymphocytic infiltration through regulating numerous cytokines produced by several cell types. Because of the widespread expression of IL-4Rα and STAT6 in various cell types,¹ both pulmonary resident cells and infiltrating cells are probably affected by the delivered genes in this model.

In summary, human IL-4 and IL-4δ2 are functionally active in mouse lung in vivo, with substantial, yet incomplete, similarity to the functional effects of corresponding species-specific IL-4 isoforms. Moreover, although previous studies in cell culture have suggested that IL-4δ2 is by itself an inactive competitive inhibitor of IL-4,^13,14,32 our current study shows IL-4δ2 in vivo to be an active mediator of lymphocytic inflammation and a Th1 cytokine pattern. The mechanistic reasons for the discrepancy between the lack of IL-4δ2 effects in cell culture and a rather pronounced functional activity of IL-4δ2 in vivo remain unclear. Together, these findings open new opportunities for pre-clinical testing of human IL-4-targeting approaches in animal models and expand our understanding of the complexity of IL-4-mediated regulation.

Disclosures

None of the authors has any conflict of interests to disclose.