Interleukin-13 and IgE production in rat experimental schistosomiasis.

ARTICLE

INTRODUCTION

IL-4 and IL-13 are considered to be key cytokines in the induction and maintenance of IgE production [1, 2]. IL-13 is produced by several cell types, such as activated T cells, dendritic cells, mast cells and alveolar macrophages [3, 4]. In contrast to IL-4, IL-13 does not act on T cells, due to the lack of expression of functional IL-13 receptors by this cell population [5, 6]. These two cytokines have many similar biological activities [6, 7]. This is due to the fact that IL-13 and IL-4 receptors share the IL-4 receptor alpha-chain, required for signal transduction [8-10]. In man and in mouse, two different cDNAs encoding IL-13 binding proteins have been cloned and are known as IL-13Ralpha1 and IL-13Ralpha2. The former is associated with IL-4Ralpha and is functional, whereas the latter binds specifically to IL-13 without inducing cell activation. IL-13Ralpha2 could thus play an important role in down-regulating the IL-13-associated effects [11].

Similarly to IL-4, IL-13 has anti-inflammatory properties and can inhibit the production of proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6 and TNF-alpha) and chemokines [12, 13]. Beside these activities, IL-13 upregulates MHC class II and CD23 expression [12]. In human B cells, IL-13 exerts similar effects to those expressed with IL-4 and can induce IgM, IgG and IgE synthesis from highly purified B cells in the presence of anti-CD40 monoclonal antibodies or activated CD4⁺ T cells [1, 2, 13-15].

The fact that IL-13 has been found to be an inducer of IgE production and therefore may contribute to the development of allergic processes, raises the question regarding the relative contribution of IL-4 and IL-13 to IgE synthesis in vivo. Such studies are hampered by the lack of suitable animal models, since bioactive IL-13 injected to mice does not lead to an IgE production in vivo or to induction of the production of this isotype by purified B cells in vitro [6, 16]. Nevertheless, the protective effect of IL-13 in the control of infection with Trichuris muris and Nippostrongylus brasiliensis has been shown in gene-targeted mice and cytokine neutralization studies in vivo [17]. Moreover, it has been suggested that IL-13 could play a more important role than IL-4 in the induction of resistance to Nippostrongylus brasiliensis [18].

In mice, the role of IL-13 in IgE production has not been elucidated and until now its direct role has remained questionable, since a specific receptor has not been characterized on murine B cells [9]. In mice injected with eggs from S. mansoni, high levels of serum IgE were reported to be induced through an IL-13-dependent pathway, since treatment of these mice with soluble IL-13R partially inhibited the expression of IgE antibodies [19]. Nevertheless, mice administered with recombinant IL-13 were found to be unable to produce significant amounts of IgE antibodies, although they produced high levels of IgG [16]. Based on these results, it was suggested that a direct interaction of IL-13 with mouse B cells could take place, although no evidence of direct binding of IL-13 or the presence of functional receptors on B lymphocytes had been presented.

In rats, the respective implication of IL-4 and IL-13 in IgE production has not yet been investigated. In previous studies, we have shown that S. mansoni infection can drive a strong Th2 response in rats with a predominant expression of IL-4 and IL-5 [20, 21]. The high levels of IgE antibodies present in sera from S. mansoni infected rats [22-25] could depend upon IL-4, produced mainly by spleen cells [21]. However, a possible role of IL-13, which has not been analyzed in these studies, could not be excluded. The purpose of our study was to investigate the expression of IL-13 and to examine its potential involvement in IgE synthesis during infection of rats with S. mansoni.

MATERIALS AND METHODS

Parasites and laboratory hosts

A Guadeloupean strain of S. mansoni was maintained in Biomphalaria glabrata snails as intermediate hosts and OF-1 mice as definitive hosts. Cercariae for experimental infections were used within 1 hour of collection.

Primary and secondary infections by S. mansoni

Male, 6-8 week-old, inbred Fischer F344 rats (Iffa Credo, France) were exposed percutaneously to 2 x 10³ cercariae (primary infection), as previously described [26] and reexposed to 2 x 10³ cercariae 63 days later (secondary infection).

Antigens

Based on the presence of soluble cross-reactive antigens between cercariae and schistosomula [21], and in addition to the fact that in experimental rat schistosomiasis, there are neither adult worms nor egg production, we used cercarial antigens in ELISA and stimulation experiments. Cercarial soluble antigens were prepared from cercariae homogenized in phosphate-buffered saline (PBS) containing 0.5 mM phenyl-methyl-sulphonyl-fluoride (PMSF) and 1 mM ethylenediamine tetra acetic acid (EDTA).

Homogenates were sonicated 6 times for 30 sec using a sonicator (Labsonic U., B. Braun, Strasbourg, France) and centrifuged twice at 10 x 10³ g for 10 min at 4° C. The soluble fraction was recovered and used as the source of antigens. The measurement of protein content was performed using the BCA protein assay reagent (Pierce, USA).

Analysis of total IgE by immunocapture ELISA

The level of total IgE was measured by ELISA (immunocapture enzyme-linked immunosorbent assays). Nunc Immunomodules (Nunc, Denmark) were coated overnight at 4° C with mAb against the epsilon heavy chain of rat IgE (MARE-1, Bazin, University of Louvain, Belgium) in 0.05 M carbonate/bicarbonate-buffer at pH 9.6 at 100 mul per well, and 5 mug/ml. After washing and blocking steps (with PBS-5% milk), samples were diluted and incubated overnight at 4° C. Plates were then incubated with biotin-labelled mAb against rat kappa and lambda light chain at 1:10,000 (MARK-1+MARL-15, Bazin, University of Louvain, Belgium) for 90 min at 37° C. After incubation for 30 min at 37° C with streptavidin-horseradish peroxidase conjugate (1:2,000) (Amersham, France), the reaction was revealed as described [21]. The amount of total IgE was evaluated by reference to standard curves constructed with known amounts of rat IgE kappa (IR 2) (Bazin, University of Louvain, Belgium).

Analysis of specific IgG by immunocapture ELISA

Specific IgG subclasses (IgG1, IgG2a, IgG2b, IgG2c) to S. mansoni cercarial antigens were measured by an ELISA as previously described (21).

Reverse transcription-PCR detection of cytokine mRNA

­ Isolation and purification of mRNA

Each tissue mRNA sample was analysed by RT-PCR separately. For tissue cytokine mRNA determinations, the right lung, spleen and liver were removed (2 rats infected and reinfected per group, 2 rats infected and non-reinfected per group and 2 rats non-infected as control) at different time points as indicated in Figure 1 and immediately frozen in liquid nitrogen. Frozen organs were homogenized in 1 ml of RNA PLUS solution (Bioprobe Systems, France). Total RNA was isolated as recommended by the manufacturer. The RNA was resuspended in RNase-free sterile water. The amount and quality of RNA were determined by spectrophotometry and analysed by agarose gel electrophoresis.

­ RT-PCR reactions

A reverse transcriptase-PCR procedure was performed to determine the relative quantities of mRNA for
IL-13 and beta-actin. Reverse transcription of RNA was performed in a final volume of 25 mul containing 0.5 mug random oligo-dT and 2.5 mug total RNA. The RT reaction and PCR reactions were performed as described in [21].

IL-13 primer sequences were 01-IL-13 (sense) : 5'- GCC CTC AGG GAG CTT ATC-3' and 02-IL-13 (anti-sense) : 5'- TGG TAT CTG GGG GGC TGG-3'. These primers were tested by PCR on genomic DNA and no fragment of the expected size was found. PCR reaction conditions were defined such that a linear relationship between input RNA and final PCR product was obtained. Both positive and negative controls were included in each assay to confirm that only cDNA PCR products were detected and that none of the reagents was contaminated with cDNA or previous PCR products. For the PCR reaction, after a first step of denaturation for 3 min at 94° C, temperature cycling was initiated 1) 94° C for 1 min, 2) 55° C for 1 min 3) 72° C for 1 min on the PTC 200 MJ Research thermal cycler (Watertown, MA, USA).

­ Analysis and quantification of PCR products

After an appropriate number of PCR cycles (28 cycles for beta-actin, IL-13), the amplified DNA was analysed by electrophoresis, Southern blotting, and hybridization with radioactive cytokine probes. Ten mul of each reaction were electrophoresed on a 1% agarose gel visualized by ethidium bromide staining and were transferred by capillary action on Hybond-N membrane (Amersham, UK) as described [5]. Membranes were then hybridized with IL-13- or beta-actin-specific oligonucleotides (gamma³²P) ATP radiolabelled (So-IL-13: 5'-TTG GTC AGG GAT TCC AGG GCT GCA CAG AAC-3'). Positive and negative controls were included in each experiment. Cytokine transcript levels were normalized to equal amounts of cDNA using the corresponding signals obtained for beta-actin RNA. Signal intensities were then compared with those obtained for samples from normal rats and results expressed as the fold increase over control. The time study experiment was repeated twice. In our study, based on absolute intensity results of cytokine transcripts obtained from 24 controls (uninfected rats), we found that the intensity varied from one to two fold. Therefore, to be in suitable confidence range, we considered significant difference between test and control when the fold increase was greater than 2.

Immunofluorescence staining of intracellular IL-13

Cytospins of freshly purified cells from spleens of S. mansoni-infected rats at days 21 and 42 were fixed in acetone at ­ 20° C for 20 min. After saturation with 10% normal mouse serum diluted in 1% PBS-BSA for 30 min at room temperature, cytospins were incubated with mAb anti-human IL-13-FITC which recognizes the rat IL-13 (Diaclone, Besançon, France) at a concentration of 25 mug/ml in 1% PBS-BSA for 90 min at room temperature in a humid chamber in the dark. After 3 final washes with 0.1% PBS-BSA and 2 with PBS, the slides were mounted with Fluoprep (Biomerieux, France) and analyzed with the fluorescence microscope. As negative control, cells were treated with FITC-isotype matched mAb (Diaclone, Besançon, France). As positive control, insect cells (Sf9) which produce rat recombinant IL-13 using a baculovirus construction, were treated according to the same protocol. The constitutive production of IL-13 was determined in cells from uninfected rats.

Lymphocyte cultures and IL-13 detection

Spleens were removed from infected or reinfected rats, and cell suspensions were prepared as followed. Briefly, spleens were forced through fine wire mesh and splenic erythrocytes were lysed by osmotic treatment (170 mM Tris-buffered saline, 155 mM ammonium chloride solution) followed by three washes in RPMI 1640 (Gibco BRL, Courbevoie, France). Cell viability was evaluated by trypan blue dye exclusion and 10⁷ cells were resuspended in 1 ml of culture medium which was RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum (JRH BioSciences, Lenexa, KS, USA), 50 mug/ml gentamycin (Schering-Plough, Levallois-Perret, France), 2 mM glutamine (Seromed, Berlin, Germany), 1% non-essential amino acids (Seromed), 1 mM sodium pyruvate (Sigma, St Louis, MO) and 5 x 10^{­ 5} M beta-mercaptoethanol (Merck, Darmstadt, Germany). One ml of cell suspension was incubated in the presence of Schistosoma mansoni cercarial soluble antigens (50 mug/ml) or Concanavalin-A (5 mug/ml, Seromed) in 24-well plates (Nunclon™, Nunc, Roskilde, Denmark) at 37° C in a 5% CO₂ atmosphere. Supernatants were collected after 48 hours for IL-13 measurements.

In order to measure IL-13 in supernatants from stimulated rat spleen cells and in sera, we used the Pelikine Compact™ human IL-13 ELISA kit (Tebu), as recommended by the manufacturer. The human IL-13 ELISA kit was checked with different rat cytokines expressed in baculovirus (IL-4, IL-5, IFN-gamma, IL-13), and rat IL-13 was specifically detected at concentrations ranging between 0.5 and 125 pg/ml (data not shown). Based on these results, we used this kit throughout our study.

Production of antiserum to rat IL-13

Recombinant rat IL-13 was produced in a bacterial expression system (Qiagen, CA). Rat IL-13 cDNA obtained by RT-PCR [27] was subcloned into pQE30 plasmid. Purified rat IL-13 was solubilized in PBS containing 0.02% SDS. A New Zealand rabbit was immunized by injection of 500 mug of recombinant IL-13 as previously described (28) in the presence of Freund's complete adjuvant (Sigma). Booster immunizations were administered by subclavicular injections of 200 mug of recombinant IL-13 in incomplete Freund's adjuvant (Sigma). Sera were collected periodically and tested for anti-IL-13 antibodies. All sera with titers > 100,000 were pooled and used for IgG purification.

Purification of polyclonal and monoclonal IgG

Rabbit antiserum to rat IL-13 was used to purify IgG according to the method using caprylic acid [29], and followed by saturated ammonium sulphate precipitation. The level of IgG purity was checked by the adsorption of IgG on protein A and was > 90%.

A mouse IgG1 anti-rat IL-4 neutralizing hybridoma cell line (OX-81) was a gift of Dr I. Anegon (Nantes, France). Cells were routinely maintained in RPMI containing 10% FCS, 1% L-Glutamine, and the hybridoma cells were injected in Balb/c mice. After 2 to 3 weeks, ascitic fluid was collected and a 50% saturated ammonium sulphate precipitation was carried out for 30 min at room temperature. The precipitate, centrifuged at 5,000 g for 15 min and dissolved on ice in PBS was dialysed three times against 5 liter volume changes of PBS (pH 7.2) for 36 hours at + 4° C. The amount of purified anti-IL-4 mAb and anti-IL-13 antibodies was measured by the BCA protein assay kit reagent (Pierce, USA) and their specificity by ELISA.

Treatment with anti-IL-4 or anti-IL-13 purified IgG antibodies

Treatments of rats with purified anti-IL-4 mAb, purified anti-IL-13 antibodies or with both were performed before and after a primary infection on days ­ 1, + 4, + 7, + 11, + 15, + 21 and before and after a secondary infection on days ­ 14, ­ 7, + 1, + 7 and + 14. Based on published data and on preliminary experiments from our laboratory, the quantities of anti-IL-4 mAb and of anti-IL-13 were 1 mg and 5 mg respectively. Administrations of anti-IL-4 mAb and/or of anti-IL-13 were done alternatively by i.p. and i.v. injections. In control groups, rats were injected with purified IgG antibodies from normal rabbit serum and/or with isotype-matched mAb.

Administration of IL-13 to S. mansoni infected rats

Endotoxin-free human recombinant IL-13 was a generous gift from C. Labbit-Lebouteiller (Sanofi, Labège, France). To assess the effect of IL-13 on total IgE production during rat schistosomiasis, a group of seven rats was injected i.p. with 1 mug of IL-13 in 1 ml of PBS on days 0 (infection day), 2, 4, 6, 8, 10, 12. The control group received PBS. Rats were bled weekly and total IgE levels were followed by ELISA as described above.

Statistical analysis

Statistical analysis was performed using Student's t-test. A value of p < 0.05 was considered as significant.

RESULTS AND DISCUSSION

IL-13 mRNA expression during primary and secondary infection of rats with S. mansoni

In a previous study, we have shown a predominant expression of IL-4 mRNA and protein in primary and secondary infected rats [20, 21]. The high levels of IgE antibodies present in sera from S. mansoni-infected rats could depend upon IL-4, produced mainly by spleen cells. However, a possible role of IL-13, which has not been analyzed in these studies, could not be excluded. To determine whether IL-13 is up-regulated, spleen, liver and lungs extracts from infected rats were assessed for IL-13 mRNA content by RT-PCR. After a primary infection, a significant increase of PCR product was detectable in the spleen on days 7 and 29 and in the liver on day 42 (Figure 1a, 1b respectively). The follow-up of IL-13 mRNA in the lungs did not show any significant expression up to 63 days, although significant levels were detected between 70 and 84 days after the primary infection. It is important to note that the magnitude of IL-13 mRNA in the lungs was high when compared to other tissues (Figure 1c).

After a secondary infection (Figure 1, hatched bars), the kinetic study of IL-13 mRNA revealed a significant increase in spleens from reinfected animals on days 84 and 91 (Figure 1a) when compared to either infected or to control animals (p < 0.05). In the liver, detectable levels were found at day 91 in reinfected rats with a significant increase when compared to infected animals (Figure 1b). A significant increase of pulmonary IL-13 mRNA in reinfected rats was clearly detectable at days 70, 74 and 91 (Figure 1c) when compared to infected animals. Therefore, during rat infection with S. mansoni, IL-13 mRNA was upregulated in spleen, liver and, more importantly, in lungs. Moreover, these data showed that IL-13 was the exclusive cytokine which is upregulated in lungs during schistosomiasis since no up-regulation of IL-4, IL-5 or IFN-gamma was observed in the same tissue [21].

IL-13 production during rat infection with S. mansoni

RT-PCR analysis demonstrated that infected rats produce IL-13 mRNA. However, these results did not prove the production of IL-13 protein in vivo. To answer this question, we examined the intracellular IL-13 protein production by freshly isolated cells and the antigen-dependent secretion of IL-13 by splenocytes.

By comparison to negative and positive controls (Figure 2a and 2b ; normal Sf9 cells and Sf9 expressing the rat recombinant IL-13 respectively), positive staining with anti-IL-13 mAb was detected by immunofluorescence staining in spleen cells purified from rats at days 21 and 42 after a primary infection (Figures 2d and 2f). Control sections with FITC labelled isotype-matched mAb did not give any positive staining (Figure 2c and 2e). No positive reactions were found with spleen cells from normal rats probed with FITC-anti-IL-13 mAb (data not shown).

The ability of spleen cells to secrete IL-13 in vitro in response to S. mansoni antigens was then investigated, the kinetic study of IL-13 release by splenocytes activated with cercarial soluble antigen showed significant release at days 21 and 42 after infection (Figure 3).

IL-13 secretion was increased at day 63 after a primary and secondary infection. IL-13 levels decreased at 74 and 78 days, increased again between 84 and 91 days. There was no significant difference between IL-13 levels secreted by spleen cells from primary and secondary infected rats (except at day 84), suggesting that they responded in a similar manner to in vitro antigen stimulation. These findings indicate that spleen cells are important sources of IL-13 following antigen stimulation. Nevertheless, it is to note that we found a high upregulation of IL-13 mRNA in lungs which could significantly contribute to the production of IL-13.

Detection of IL-13 in sera from rats infected with S. mansoni

To ascertain whether cells would directly secrete
IL-13 in vivo, an assessment of IL-13 levels in sera from infected rats was made. One representative experiment out of two (using 7 rats at each timepoint and in each experiment) is shown in Figure 4. Low but significant levels were detected in sera from infected rats when compared to those of control rats (< 0.5 pg/ml). Importantly, as shown in Figure 4, maximum levels were found 3 weeks post infection. However, these levels did not parallel the secretion of IL-13 by activated spleen cells in vitro observed sometime after a primary infection and soon after a second. It is suggested from these data, that the circulating IL-13 does not necessarily reflect the level of IL-13 produced locally in different tissues of infected rats. It is to note that IL-13 is not only produced by Th2 cells but also by mast cells and human alveolar macrophages [4, 30]. These observations in addition to those showing that mast cells from S. mansoni-infected rats were activated by immunological stimuli (IgE and specific antigens), as assessed by the release of mast protease [22], suggest that the source of IL-13 during experimental rat schistosomiasis might be lymphoid cells and mast cells.

Effects of anti-IL-4 and anti-IL-13 antibodies on IgE production in vivo

From the above data and our previous studies, it can be concluded that rats produce IL-4 and IL-13 during schistosomiasis which is accompanied by increased IgE levels. In order to know whether both IL-4 and IL-13 are involved in the IgE response, as in humans, rats were treated with anti-IL-4 and/or anti-IL-13 antibodies. Administration of anti-IL-4 antibodies repeatedly before and during infection induced a slight decrease of IgE levels but this did not achieve statistical significance (Figure 5a). Rats treated with anti-IL-13 antibodies alone showed no significant decrease of IgE levels when compared to animals treated with control antibodies (Figure 5b). These results indicate that a compensatory mechanism, provided either by IL-4 or IL-13, exists to maintain IgE levels. When both anti-IL-4 and anti-IL-13 antibodies were co-administered, a significant decrease in IgE antibodies at day 21 after a primary infection was observed when compared to infected animals treated with control antibodies (Figure 5c). However, the follow up of IgG1, IgG2a, IgG2b and IgG2c antibodies did not show significant decrease in cotreated rats when compared to control animals (data not shown). The inhibitory effect of cotreatement by anti-IL-4 and anti-IL-13 antibodies on IgE levels appeared of a brief duration since the follow up thereafter did not show any significant decrease. Similarly, after a secondary infection, no significant decrease in IgE levels was observed (data not shown). A possible explanation could be an enhanced secretion of IL-13 and IL-4 by activated T cells and mast cells. This may be due to a considerable cell activation by an extensive release of schistosome antigens during the expulsion of the first and the second worm population which occurred within 28 days after a primary infection and 14 days after a secondary infection respectively [21]. It seems therefore that, under the experimental conditions used throughout this work, the production of IgE antibodies could be inhibited at least early after infection (up to 21 days after the primary infection) and before worm expulsion, by co-neutralization of IL-4 and IL-13.

Effect of exogenous IL-13 on IgE production in vivo

To assess the effect of IL-13 administration on IgE production in vivo during schistosomiasis, rats were treated with i.p. injection of IL-13, at 2 day intervals, from the day of infection to day 12 after infection. Circulating IgE levels were followed up in a kinetic study. Figure 6 shows that the treatment of rats with IL-13 resulted in an enhanced production of IgE antibodies and led to a significant increase at days 28 (52% increase), 35 (105% increase) and 42 (83% increase) post-infection when compared to the control group. These results are not in agreement with those obtained in mice where IL-13 treatment did not induce IgE production [16]. However, they are in accordance with the in vitro IL-13 effect on the induction of IgE synthesis by human B cells [2]. It is important to note that the appearence of the IgE antibodies was not accelerated in IL-13-treated rats when compared to untreated animals. This might be explained by the fact that the IL-13 requires an earlier activation of the immune system by another cytokine, probably IL-4, in order to potentiate and maintain IgE synthesis, as was suggested in humans [6]. Whether the in vivo effect on IgE could have been due to direct or indirect effects of IL-13 can not be answered so far.

Taken together, data presented above show that, similarly to IL-4, IL-13 is upregulated and is secreted during rat schistosomiasis, suggesting a possible involvement of both cytokines in IgE induction. In the in vivo experiments, rats co-treated with neutralizing anti-IL-4 and anti-IL-13 antibodies showed significant decreases in the IgE levels suggesting the contribution of both IL-4 and IL-13 in the induction of IgE response. Thus, the rat provides a relevant model for the better understanding of the implication of IL-13 in IgE synthesis in humans, where it has been shown that IL-13 is involved by acting directly at the B cell level.

CONCLUSION

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