IL-10 regulates early IL-12-mediated immune responses induced by the radiation-attenuated schistosome vaccine

Karen G. Hogg1, Supeecha Kumkate1 and Adrian P. Mountford1

1 Department of Biology (Area 5), PO Box 373, University of York, York YO10 5YW, UK

Correspondence to: A. P. Mountford; E-mail: apm10{at}york.ac.uk
Transmitting editor: A. Kelso


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Radiation-attenuated (RA) schistosomes penetrate the host via the skin where they stimulate intense inflammatory reactions and the release of pro-inflammatory IL-12, important for Th1-type immune responses which are partially host protective. However, RA larvae also induce the secretion of regulatory IL-10. We now show that following vaccination of IL-12p40–/– mice, abundant IL-10 was produced by in vitro cultured skin biopsies between days 4 and 14, corresponding to the down-regulation of MHC II expression by cells in the dermis and cells that emigrate from the skin. In IL-10–/– mice, inflammation of the vaccination site was increased with larger numbers of IL-12p40+, MHC II+ and CD86+ cells in the dermal exudate, and was associated with elevated levels of skin-derived IL-12p40 and IL-1ß. These changes in IL-10–/– mice were also reflected by an increased number of cells in the skin-draining lymph nodes (sdLN) and greater levels of lymphocyte proliferation. Moreover, such mice had increased numbers of CD4+ sdLN cells that were CD25+, CD28+ or CD152+ and accessory cells that were CD40+ or MHC II+. Finally, the secretion of IFN-{gamma} (and IL-12p40) by in vitro cultured sdLN cells was substantially raised in IL-10–/– mice, but much reduced in IL-12p40–/– mice, resulting in the development of highly polarized Th1 and Th2 cytokine profiles in the two groups of mice respectively. We conclude that IL-10 has an important role early in the regulation of IL-12-mediated priming of acquired immune responses, and effectively contains excessive dermal inflammation and prevents the development of highly polarized Th1-type responses.

Keywords: helminth, innate, parasite, skin, Th1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The skin is an important organ of the innate immune system, forming an initial line of host defence against infectious pathogens. Immune events in the skin are also likely to be critical in guiding the subsequent priming of adaptive immune responses, particularly in the skin-draining lymph nodes (sdLN), which can confer protective immunity to re-infection. In this context, schistosomes are parasitic helminths that infect mammalian hosts via the skin. Free-swimming cercariae in contaminated water make contact with the skin and burrow through the different layers aided by an array of proteases (1). Following maturation, schistosomes cause a serious debilitating disease, which affects >500 million people worldwide, and although drug-treatment can be effective, there is a pressing need to develop an efficient vaccine.

In the murine model of infection, normal parasites are not thought to induce significant levels of immunologically mediated protection against challenge infection (2,3). They are also inefficient stimulators of innate immune responses in the skin (4), and acquired immune responses in the sdLN (5) and lungs (6). In contrast, cercariae attenuated with optimum doses of irradiation consistently induce CD4+-dependent protective immunity of between 60 and 70% (7,8). Acquired immune responses to radiation-attenuated (RA) larvae have been well characterized, demonstrating a strong bias towards Th1-type, IFN-{gamma}-mediated protection (5,9), although a role for antibodies is also evident (1012).

Priming of an efficient acquired immune response is dependent upon the generation of a polarized Th1-type immune response in the sdLN within the first 7–14 days after vaccination (5,13), followed by recruitment of these cells to the lungs (14). However, it is less clear what immune-related events occur in the skin in the first 2 weeks which will contribute towards immune priming in the sdLN. One key feature of RA larvae is that they are much slower to migrate through the skin, thereby extending the period over which they can present antigens to accessory cells of the innate immune system (4,15,16). We recently reported that RA larvae were efficient stimulators of various cytokines and chemokines in the skin immediately after vaccination, leading to a strong cellular inflammatory response (4). Chief amongst the cytokines was sustained IL-12p40 induced by RA, but not normal, larvae. IL-12 is a highly potent cytokine able to stimulate NK and Th1 cells to produce IFN-{gamma} (17), but it also activates potential antigen-presenting cells (APC) such as macrophages and dendritic cells (DC) (18). Indeed, IL-12 has previously been shown to be important in the development of Th1-mediated immunity (19), since vaccinated IL-12p40–/– mice have substantially reduced levels of protection against Schistosoma mansoni (~30–40%) that can be restored by intradermal administration of recombinant IL-12 (20). Nevertheless, we also report that significant quantities of immunoregulatory IL-10 are produced in the skin in response RA larvae (4). This cytokine is likely to have a major role in down-regulating immune responses in the skin and the sdLN by acting on those accessory cells that conventionally secrete IL-12 (21). Indeed, in the absence of IL-10, mice exposed to two doses of RA larvae develop a more polarized Th1-type cytokine profile and exhibit high levels of protective immunity of >90% (10).

In light of the observation that both IL-12 and IL-10 are produced in the skin soon after exposure to RA schistosome larvae, we wished to examine their relative roles during the development of inflammatory responses in the skin and the priming of Th cells in the sdLN. We report here that IL-12 and IL-10 act as antagonistics regulating cytokine production in the skin and the expression of MHC II by skin-derived accessory cells. This in turn affects the efficiency of immune priming in the sdLN where the absence of IL-10 leads to increased co-stimulatory molecule expression and the enhanced production of Th1-associated cytokines. The early expression of IL-10 following exposure to the RA schistosome vaccine has important implications in the design of vaccination strategies against different pathogens where a strong Th1 response is required, but also where excessive inflammation needs to be controlled.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Parasite and host
Infective cercariae of S. mansoni were attenuated with 20-krad irradiation (60Co source), and then used to vaccinate groups of anaesthetized female IL-12p40–/– (22) and IL-10–/– mice (23) (Taconic Farms, Rockville, MD) alongside wild-type C57BL/6 strain cohorts via each pinna as described previously (4,12).

Sampling regime
At various time points (days 0, 1, 2, 4, 8 and 14) post-vaccination (p.v.), groups of mice (providing 4–6 pinnae) were sacrificed and the extent of inflammation at the exposure site determined by measurement of the pinnae thickness using a dial gauge micrometer (Mitutoyo, Kawasaki, Japan). The pinnae and the auricular draining sdLN were then removed and prepared for histology, culture in vitro or analysis of surface marker and co-stimulatory molecule expression by flow cytometry.

Histology
Pinnae were fixed in 10% neutral buffered formalin (Sigma, Poole, UK), wax-embedded, sectioned at 6 µm and stained with H & E (EHL, Warrington, UK). For immunohistochemistry, 12-µm cryosections were fixed in 3.7% paraformaldehyde in PBS for 20 min, washed and air dried. Sections were blocked first with 3% BSA, and then with 1% H2O2 in PBS for 15 min and biotin blocking kit (Vector, Peterborough, UK). Sections were then incubated with biotinylated anti-MHC II antibody (B21.2, Iab,d; Caltag-MedSystems, Towcester, UK) for 30 min at room temperature, followed by Vectastain Elite ABC peroxidase reagent combined with Vector VIP enzyme substrate and counterstained with methyl green (Vector).

Preparation of pinnae and sdLN cells for in vitro culture and flow cytometry
Pinnae were cultured in vitro as described previously (12). Briefly, each pinna was split and the two faces floated on 0.5 ml RPMI medium containing 10% FCS (low endotoxin; Seralab, Kidlington, UK), 2 mM L-glutamine, 200 U/ml penicillin and 100 µg/ml streptomycin (Sigma; RPMI/10) in 24-well hydrophobic culture plates (Greiner Labortechnik, Fricken hausen, Germany). Pinnae were cultured in the absence of any added parasite antigen for 18 h at 37°C, 5% CO2. The culture supernatant was then collected, pooled from the two faces for each pinna and stored at –20°C for subsequent cytokine analysis. The cell population which had detached from the dermis was collected and resuspended in phenotyping buffer (PBS + 0.01% CaCl2, 0.01% MgCl2 and 0.1% BSA) prior to labelling and analysis by flow cytometry. For intracellular staining of IL-12p40, cells were cultured as above, but in the presence of 1 µg/ml GolgiPlug (BD PharMingen, Oxford, UK) for 12 h to block cytokine secretion.

The sdLN were removed and disrupted to produce a single-cell suspension (12,14). For analysis by flow cytometry, sdLN cells were resuspended in phenotyping buffer as above. For in vitro culture, cells were resuspended in RPMI/10 and cultured in 96-well flat-bottomed plates at a concentration of 4 x 106/ml in the presence, or absence, of 50 µg/ml soluble larval antigen preparation (SSP) (24) for 72 h at 37°C, 5% CO2. Culture supernatants were removed and stored at –20°C. For the detection of intracellular IFN-{gamma}, sdLN cells were cultured in the presence of 1 µg/ml plate-bound {alpha}CD3 antibody (BD PharMingen) for 72 h and then for 6 h in the presence of 1 µg/ml GolgiPlug (BD PharMingen). Proliferation of sdLN cells following 4 days in vitro culture in the presence of SSP was determined following the addition of 0.5 µCi/well [3H]thymidine (Amersham Biosciences, Little Chalfont, UK) during the last 18 h. Cells were then harvested and the incorporation of isotope into cellular DNA determined by scintillation counting (TopCount; Packard, Pangbourne, UK).

Cytokine and ELISAs
Specific cytokines released into the culture media from split pinnae and from sdLN cell suspensions were quantified using double-antibody ELISAs as detailed previously (4,12). The lower levels of detection in these ELISAs were 20 pg/ml for IL-12p40, IL-6 and IL-4, 25 pg/ml for IFN-{gamma}, IL-1ß and IL-10, and 100 pg/ml IL-18. All supernatants were tested neat. Data comparisons were tested for significance by using Student’s t-test. Arithmetic means are shown ± SEM. All experiments were repeated between 2 and 4 times.

Flow cytometric analysis
Single-cell suspensions of dermal exudate and sdLN cells were labelled with anti-mouse mAb prior to analysis by two-colour flow cytometry (Coulter XL; Coulter, Luton, UK). Briefly, 50 µl aliquots of up to 5 x 105 cells were blocked with 0.5 µg anti-CD16/CD32 (BD PharMingen) and incubated in optimal dilutions of mAb for 30 min on ice. Antibodies were as follows: MHC class II, anti-Iab,d (M5/114.15.2; BD PharMingen), CD4 (L3/T4; Caltag), CD25 (PC61 5.3; Caltag), CD28 (37.51.1; Caltag), CD40 (3/23; BD PharMingen), CD86 (RMMP-2; Caltag), CD152 (UC10-4F10-11; BD PharMingen) and CD154 (39H5; Serotec). Antibodies were either directly labelled with fluorochromes (FITC or phycoerythrin) or biotin, in which case streptavidin–Quantum Red (Sigma) was used as a detection probe. Irrelevant isotype-matched antibodies from the relevant supplier were used as controls throughout. For intracellular staining of CD152, IL-12p40 and IFN-{gamma}, cells were treated with Cytofix/Cytoperm (BD PharMingen) prior to and during incubation steps. Anti-cytokine mAb were phycoerythrin-labelled anti-IFN-{gamma} (XMG1.2; Caltag) or anti-IL-12p40 mAb (C15.6; BD PharMingen), or rat IgG1 isotype control.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Inflammation of the skin is greater in the absence of IL-10
After vaccination, inflammation of the exposure site as judged by pinnae thickness was significantly increased in all three groups of mice by day 2 (all P < 0.05) compared to their naive cohorts, which were all very similar in size (Fig. 1A). In wild-type mice, the peak of inflammation was reached by day 4, after which inflammation declined to near naive levels by day 14. A similar profile was observed for IL-12p40–/– mice which were also significantly less inflamed than wild-type mice on days 2 and 4 (P < 0.05 and P < 0.01 respectively). In contrast, inflammation of pinnae from vaccinated IL-10–/– mice continued to increase from day 2 out to day 14. Indeed, pinnae from these mice were thicker than wild-type cohorts at each time point, particularly on days 8 and 14 (P < 0.001). Pinnae from vaccinated IL-10–/– mice were also always greater than in IL-12p40–/– mice. On day 14, inflammation of pinnae from IL-10–/– mice was 47 and 67% greater than in wild-type and IL-12p40–/– mice respectively.



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Fig. 1. Inflammation of the dermal site of vaccination is greatly increased in the absence of IL-10. (A) Pinnae thickness at autopsy on days 0 (naive), 2, 4, 8 and 14 p.v in wild-type (black bars), IL-12p40–/– (patterned bars) and IL-10–/– (hatched bars) mice; data are means + SEM for 6–8 pinnae. Statistical significance is for cytokine-deficient mice in relation to wild-type mice. (B) Micrographs show the extent of dermal inflammation and cellular infiltrate through traverse sections of pinnae stained with H & E obtained from the three groups of mice on day 14 p.v. Naive tissue from wild-type mice is shown for comparison. Scale bar = 50 µm.

 
The profile of dermal inflammation in the different groups judged by pinnae thickness was also observed by histological examination (Fig. 1B). Although sample preparation leads to shrinkage, it can be seen that the pinnae sections of IL-10–/– mice are still substantially thicker than wild-type or IL-12p40–/– mice at day 14. In all groups of mice there is evidence of a substantial cellular influx (not edema) into the dermis compared to naive mice (Fig. 1B), but the cellular infiltrate in IL-10–/– mice was by far the most intense, resulting in a much thicker dermis. Detailed immunohistochemical analysis revealed that the cellular influx was comprised of granulocytes (Gr-1+), neutrophils (7/4+), macrophages (CD11b+ and F4/80+) and DC (CD11c+) (Kumkate et al., manuscript in preparation).

Patterns of cytokine produced by skin biopsies are deregulated in IL-12p40–/– and IL-10–/– mice
The cytokine profile in IL-12p40–/– and IL-10–/– mice after vaccination was compared in order to seek an explanation for the changes seen in the profiles of dermal inflammation and cellularity. As expected, IL-12p40 was not detectable in the supernatants of in vitro cultured pinnae biopsies obtained from IL-12p40–/– vaccinated mice, although high levels were produced by pinnae from wild-type mice, and these were maintained at days 2, 4, 8 and 14 p.v. (Fig. 2A). IL-12p40 was also produced in abundance by the pinnae from IL-10–/– mice; the levels were significantly greater than in wild-type mice at all times (P < 0.01–0.001) and were up to 2-fold greater on day 14. IL-10 was produced by pinnae from wild-type mice at all time points, albeit at slightly lower levels on days 8 and 14 (Fig. 2B). However, production of this cytokine was much greater in IL-12p40–/– mice on days 4 onwards (P < 0.001), reaching a plateau between days 8 and 14, when >9-fold more IL-10 was produced by the pinnae of IL-12p40–/– compared to wild-type mice.



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Fig. 2. Deregulation of IL-12p40 and IL-10 production in the skin of cytokine-deficient mice. Production of (A) IL-12p40, (B) IL-10, (C) IL-18 and (D) IL-1ß by in vitro cultured pinnae biopsies at 18 h in the absence of added antigen obtained on days 2, 4, 8 and 14 p.v. Bars represent wild-type (black), IL-12p40–/– (patterned) and IL-10–/– (hatched) mice; data are means + SEM for 6–8 pinnae. Statistical significance is for cytokine-deficient mice in relation to wild-type mice. The horizontal dashed line shows the level of cytokine production in naive mice, which does not differ substantially between the three groups of mice (except for IL-12p40 and IL-10 in the corresponding group of cytokine-deficient mice where the histogram bars represent the lower level of detection).

 
The substantial changes in the quantities of IL-12p40 and IL-10 secreted by the pinnae of IL-10–/– and IL-12p40–/– mice respectively were not mirrored in the pattern of IL-18 production (Fig. 2C). Indeed, although there was a 2-fold increase in the amount of IL-18 detected by day 2 p.v., there was no difference between the three groups of mice. A further increase in IL-18 was detected by day 4 p.v. and although less was produced by the samples from IL-10–/– mice, this was not significant (P > 0.05). The production of IL-1ß by pinnae from both wild-type and IL-12p40–/– mice peaked on day 2 p.v. and declined to, or below, naive levels thereafter (Fig. 2D). While a similar pattern of IL-1ß production was observed in IL-10–/– mice, the levels were greater than in the other two groups of mice. Levels of IL-1ß, IL-12p40, IL-18 and IL-10 production in naive mice for each group of mice were not markedly different, and for clarity the mean values are shown as a single horizontal line. We also detected elevated IL-6, IL-4 and IFN-{gamma} in the culture supernatants of vaccinated pinnae (data not shown). No differences were observed between the three groups of mice for the production of IL-6 and IL-4, and although peak levels of IFN-{gamma} were 3-fold greater for IL-10–/– mice (P < 0.01), these were much lower (maximum 300 pg/ml) than those seen for sdLN cultures (cf. Fig. 7).



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Fig. 7. Cytokine production in the sdLN is polarized towards the Th2 or Th1 poles in the absence of IL-12p40 and IL-10 respectively. The production of (A) IL-12p40 by sdLN cells cultured in vitro for 72 h in the absence of added antigen. The production of (B) IFN-{gamma}, (C) IL-4 and (D) IL-10 by sdLN cultured in the presence of SSP for 72 h. Intracellular IFN-{gamma} (E) detected in CD4+ cells obtained on days 4 and 14 p.v. stimulated with plate-bound {alpha}CD3 antibody. Bars represent the mean + SEM for 4–5 sdLN from wild-type (black), IL-12p40–/– (patterned) and IL-10–/– (hatched) mice.

 
In the absence of IL-12, the expression of MHC II is markedly reduced.
In order to establish what effect the production of IL-12 in the pinna had on the ability of accessory cells to act as APC, we examined the expression of MHC II in cryosections of the pinnae. By day 4 p.v., MHC II+ cells were abundant in wild-type mice, particularly within foci of cellular infiltrate in the dermis which formed in response to migrating larvae (Fig. 3B). The number of MHC II+ cells in the dermis of IL-10–/– mice was greater than in wild-type mice, but it is difficult to accurately quantify any increase (Fig. 3C) and our results could represent an increase in the level of MHC II antigen expression per cell. However, MHC II+ cells were clearly much less abundant in IL-12p40–/– mice (Fig. 3D).



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Fig. 3. Expression of MHC II is reduced in the absence of IL-12p40. Micrographs of MHC II positive staining (VIP) shown as black cells (arrowed) in pinna sections from wild-type (B), IL-10–/– (C) and IL-12p40–/– (D) mice on day 4 p.v. The pattern of staining with an isotype control antibody (A) is shown for pinna tissue from wild-type mice on day 4 p.v. Scale bar = 50 µm.

 
To be effective as APC, the MHC II+ cells need to traffic to the sdLN in order to prime CD4+ cells located in the paracortical areas. In this context, a large number of cells spontaneously emigrate from pinnae biopsies cultured in vitro peaking on day 4 (12). The number of emigrant cells was 15–25% greater for IL-10–/– mice (data not shown) and a significant proportion were found to be MHC II+ (Fig. 4A). The percentage positive for MHC II was significantly greater in IL-10–/– mice, but was lower in IL-12p40–/– mice relative to the number in wild-type mice (P < 0.05; Fig. 4A). This pattern was also observed when analysis was restricted to those cells expressing high levels of MHC II. The dermal exudate cells from IL-10–/– mice also expressed significantly higher levels of CD86 than either wild-type or IL-12p40–/– mice (P < 0.001). It was also evident that the proportion of IL-12p40+ cells in the dermal exudate was greater in IL-10–/– mice (7.17%) compared to wild-type mice (6.05%) (Fig. 4B). Moreover, >90% of all IL-12p40+ cells in the dermal exudate of IL-10–/– and wild-type mice were also MHC II+.



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Fig. 4. Dermal emigrant cells express higher levels of MHC II, CD86 and IL-12p40 in the absence of IL-10. (A) The percent of cells in the dermal exudate population positive for MHC II and CD86 as determined by flow cytometry. Bars represent wild-type (black), IL-12p40–/– (patterned) and IL-10–/– (hatched) mice; data are means + SEM for 6–8 pinnae. Statistical significance is for cytokine-deficient mice in relation to wild-type mice. (B) Staining for IL-12p40 in the dermal emigrant population from wild-type, IL-12p40–/– and IL-10–/– mice. The grey line shows staining by an isotype-matched control antibody and the black line shows staining with phycoerythrin-labelled anti-IL-12p40. Number is mean percent of cells positive for IL-12p40 compared to isotype control.

 
The absence of IL-10 leads to increased cellularity and proliferation in the sdLN
In naive mice of each strain, there was little difference between the total numbers of cells and the proportions of CD4+ and B220+ cells (Fig. 5A–C). Exposure of mice to RA larvae caused an increase in the sdLN cell number, particularly of B220+ cells which, as noted previously (25), results from an influx of naive B cells and effectively diluting the CD4+ cell population. Nevertheless, the absence of IL-10 caused a greater increase in the overall cellularity, most noticeably on day 14 when there was a 66% increase in the cell number of the sdLN from IL-10–/– compared to wild-type mice (P < 0.001; Fig. 5A). At the same time the sdLN of IL-12p40–/– mice had significantly fewer cells (P < 0.05). However, IL-10–/– mice had a significantly lower proportion of CD4+ cells on both days 4 and 14 p.v. than either of the other two groups (Fig. 5B). Indeed, on day 14, IL-12p40–/– mice had a significantly higher proportion of CD4+ cells compared to wild-type mice. In contrast, the proportion of B220+ lymphocytes in the sdLN was significantly greater in IL-10–/– mice than the other two groups (Fig. 5C). Analysis of sdLN cells cultured in vitro with soluble schistosome antigen revealed that proliferation of cells from IL-12p40–/– mice was lower than in wild-type mice on both days 4 and 14 (P < 0.01; Fig. 5D). On the other hand, the proliferation of sdLN cells for IL-10–/– mice was greater than for wild-type mice on both days 4 and 14, although the increase at the earlier time point was not significant (Fig. 5E).



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Fig. 5. Changes in the lymphocyte composition and reactivity in the sdLN in the absence of IL-10 and IL-12p40. The total number of cells (A), and the percentage of CD4+ and B220+ cells in the sdLN (B and C) for naive mice and on days 4 and 14 p.v. Bars represent wild-type (black), IL-12p40–/– (patterned) and IL-10–/– (hatched) mice; data are means + SEM for 4–5 sdLN. The proliferation of sdLN cells in response to stimulation with SSP in IL-12p40–/– (D) and IL-10–/– (E) mice as judged by the incorporation of [3H]thymidine; means are for cells from four individual mice + SEM. Statistical significance is for cytokine-deficient mice in relation to wild-type mice.

 
Markers of activation and co-stimulation are elevated in the absence of IL-10
Since levels of lymphocyte proliferation were greater in the absence of IL-10, changes in the expression of various markers of activation and co-stimulation following vaccination were investigated. In nearly all cases, differences in the levels of expression between different groups were not evident on day 4, but were readily observed on day 14. CD25 expression correlated with the pattern of lymphocyte proliferation on day 14 with elevated levels on CD4+ cells from IL-10–/– mice, but reduced levels were recorded on cells from IL-12p40–/– mice (P < 0.01; Fig. 6A). Increased co-stimulatory molecule expression on CD4+ cells was only evident for CD28 and CD152 at day 14, with no change in CD154 (Fig. 6B–D). There were no significant changes in APC expression of CD86 (Fig. 6E), but there were small significant increases in the levels of both CD40 and MHC IIhi in IL-10–/– mice (Fig. 6F–G). CD40 expression was also lower in IL-12p40–/– (P < 0.05) compared to wild-type mice, but MHC II expression was not significantly different. In all cases, statistical difference shown in Fig. 6 is for cytokine-deficient mice compared to wild-type cohorts. However, in most cases equivalent, or higher, levels of significance are obtained when comparing the data collected for IL-10–/– versus IL-12p40–/– mice (data not shown). No significant differences were recorded between the three groups of naive mice in their expression of the various co-factors described above.



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Fig. 6. Markers of lymphocyte activity and co-stimulatory molecules are up-regulated in the absence of IL-10. The percentage of CD4+ sdLN cells positive for CD25, CD28, CD154 and CD152 (A–D), and the percentage of all sdLN cells positive for CD86, CD40 and MHC IIhi (E–G). Bars represent the mean + SEM for 4–5 sdLN from wild-type (black), IL-12p40–/– (patterned) and IL-10–/– (hatched) mice. No differences between the levels of expression of any marker were detected in the three groups of naive mice.

 
The profile of cytokines produced by sdLN cells from IL-12p40–/– and IL-10–/– reveals polarization towards Th2- and Th1-type responses respectively
Cells from the sdLN of wild-type mice cultured in vitro in the absence of added antigen were efficient producers of IL-12p40 at both days 4 and 14 p.v. (Fig. 7A). In this situation, detection of IL-12 represents its production in vivo in response to antigen from the RA larvae. As expected, detection of this molecule in IL-12p40–/– mice was at or below the lower level of detection for this ELISA. In contrast, the production of IL-12p40 in IL-10–/– mice was greatly elevated above that in wild-type mice, although in this experiment significance was achieved on day 4 only (P < 0.01). Cells from wild-type mice secreted sustained quantities of antigen-driven IFN-{gamma} on days 4 and 14 p.v., but the amount produced by cells from IL-12p40–/– mice was negligible (Fig. 7B). However, the secretion of IFN-{gamma} was much greater in IL-10–/– than wild-type and IL-12p40–/– mice at both days 4 and 14 (P < 0.01). Although cells from wild-type mice produced only small quantities of IL-4, cells from IL-12p40–/– mice produced abundant IL-4, particularly at day 4 (P < 0.001; Fig. 7C). In contrast, we were unable to detect the secretion of IL-4 from the cells of IL-10–/– mice. Antigen-driven IL-10 production was greater in IL-12p40–/– compared to wild-type mice and the levels were higher at day 4 than 14. No IFN-{gamma}, IL-4 or IL-10 was detected in the absence of added antigen at either time point and none were detected in cultures of sdLN cells from naive mice. Further analysis of IFN-{gamma} production by intracellular cytokine staining shows that on day 4 there are >80% more CD4+IFN-{gamma}+ cells in the sdLN of IL-10–/– than wild-type mice, but there were nearly 60% fewer CD4+IFN-{gamma}+ cells in IL-12p40–/– mice (Fig. 7E). Moreover, the mean intensity of fluorescence for IFN-{gamma} staining was 30% greater (2.44 ± 0.32) for CD4+ cells on day 4 p.v. from IL-10–/– mice compared to wild-type mice (1.86 ± 0.24). By day 14, a similar profile was observed with 70% more CD4+IFN-{gamma}+ cells in the sdLN of IL-10–/– than wild-type mice, although the overall number of CD4+IFN-{gamma}+ cells was lower at day 14 than at day 4 in all groups of mice. Together these results confirm that the IL-10–/– mice have a more Th1-biased acquired immune response, whilst IL-12p40–/– mice are more Th2 biased.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of IL-10 is a key agent in the control of excessive inflammation and immune-related immunopathologies. In this paper, we show that the early production of IL-10 in the skin of mice exposed to the RA schistosome vaccine has an important role in controlling a series of IL-12-mediated immune events that in turn govern the scale of the ensuing Th1-type protective immune response. The initial inflammatory response in the skin is characterized by an increase in tissue thickness caused by an influx of polymorphonuclear and mononuclear cells [(4) and Kumkate, manuscript in preparation], and the rapid release of pro-inflammatory and chemotactic cytokines including IL-1ß IL-6, IL-12, MIP-1{alpha} and MIP-1ß [here and (4)]. However, we also recorded the production of IL-10 in dermal tissues, which is known to have anti-inflammatory effects, particularly in the regulation of IL-12-mediated immune responsiveness (21). This raises the question as to how IL-12 and IL-10 interact during stimulation of the innate response, and how this affects the acquired immune response.

Inflammation of skin exposed to the RA vaccine was significantly elevated in the absence of IL-10, but decreased in the absence of IL-12. Histological analysis of pinnae demonstrated that the maintenance of dermal inflammation was largely due to the presence of cellular infiltrates (rather than simply edema), while in wild-type and IL-12p40–/– mice the foci largely resolved by day 14 and resembled the pinnae of naive mice despite the predicted persistence of up to 25% of the RA larvae (15). The resolution of dermal inflammation (except in IL-10–/– mice) in the face of a substantial parasite antigen load strongly implicates an immunoregulatory role for IL-10. In fact, endogenous IL-10, but not IL-4, is a key factor in the control of dermal inflammation due to croton oil-induced contact sensitivity (26). After exposure to normal larvae, the secretion of IL-10 in the skin in response to parasite- and/or host-derived prostaglandin E2 may explain the ability of normal larvae to modulate dermal inflammation (27). Prostaglandin E2 is a potent stimulant of IL-10 production (28,29) operating via a cyclooxygenase 2-dependent pathway (30). However, our demonstration that RA larvae also induce IL-10 contradicts previous reports showing that RA larvae do not elicit IL-10 in the skin (27,31). Differences in protocol may explain our contrasting results, but we believe that RA larvae stimulate IL-10 production in the skin via a pathway which is similar, if not identical, to that proposed for normal larvae.

IL-10 appears to regulate the effects of IL-12 and IL-1ß since in its absence elevated levels of both cytokines were evident compared to wild-type cohorts. In this context, IL-10 is a potent inhibitor of IL-12 transcription (32) and interferes with IL-12 production by DC (33). As a corollary, we observed increased levels of IL-10 in IL-12p40–/– mice, demonstrating that IL-12 ordinarily restrains the production of IL-10. Such divergent patterns of cytokine production were not evident for all molecules because no differences in IL-18 (or IL-6) production between the groups were seen at any time point. Therefore, although IL-18 can act in synergy with IL-12 (34), it does not appear to be an important factor in this model. On the other hand, since IL-12 is also a potent inducer of IFN-{gamma} (18), it was not surprising that elevated levels of IFN-{gamma} were detected for IL-10–/– skin biopsies. However, only very limited quantities were detected (cf. levels in sdLN). Moreover, levels of IL-10 and IL-12 were not substantially affected by the absence of IFN-{gamma} signalling in vaccinated IFN-{gamma}R–/– mice (Hogg, unpublished observations), suggesting that IFN-{gamma} does not have an important regulatory effect upon these cytokines in the skin.

A major event in the skin following vaccination is the uptake of parasite antigen by local APC prior their migration to the sdLN where they can present antigen. In this context, IL-10 down-regulates a number of essential factors including the expression of MHC II and B7 molecules, (35,36), cytokine production (33), and the emigration of MHC II+ cells from sites of dermal inflammation (37). Conversely, IL-12 causes the up-regulation of pro-inflammatory cytokines and MHC II (38), and synergizes with B7/CD28 to promote CD4+ cell proliferation and cytokine production (39). Therefore, the presence of IL-10 and/or IL-12 should have important effects upon T cell priming through their actions on APC or antigen presentation by APC. Our data support this contention in as much that in the absence of IL-12p40, there was a decrease in the number of dermal MHC II+ cells, while the absence of IL-10 leads to an increase. Increased CD86 expression by dermal exudate cells from IL-10–/– mice is also consistent with the IL-10-mediated regulation of CD86 on dermal DC from psoriatic skin (40).

Although more cells were observed in the dermal exudate of IL-10–/– mice, in agreement with enhanced Langerhans cell migration in the absence of IL-10 (37), this may simply reflect the greater numbers in the inflamed pinnae. Moreover, in another model of schistosome-induced Langerhans cell migration, a role for IL-10 was not found (41). Nevertheless, our data strongly suggest that abundant IL-12 in the absence of IL-10 leads to an environment more conducive to the development of antigen-primed MHC II+ APC in the skin which should have clear effects upon acquired immune events and the sdLN. The absence of IL-10 resulted in greater numbers of sdLN cells on day 14, whilst antigen-driven cell proliferation was greater, supportive of findings by Hoffmann et al. (10). However, CD4+ cells were less abundant and B cells more frequent in IL-10–/– mice, suggesting that the increased sdLN cell number in these mice may be caused by a preferential expansion of B cells. In turn, this would provide conditions for increased antibody production that is a feature of IL-10–/– mice (10).

The greater levels of cell proliferation in the absence of IL-10 were reflected in slightly enhanced expression of various co-factors on accessory and CD4+ cells on day 14. Indeed, expression of CD25 is indicative of recent immune activation, while CD152 at low levels of expression has activation properties, although both have been linked to a ‘regulatory’ phenotype (42). Given the large changes in cytokine production detected in the sdLN, it is perhaps surprising that changes in the levels of activation markers and co-stimulatory molecules were not greater. Nevertheless, the general pattern supports the notion that IL-10 down-regulates the efficiency of immune priming in the sdLN.

Finally, the changes noted above in the absence of IL-10, or IL-12p40, have major effects upon the cytokine profile by cells from the sdLN. Cells spontaneously secreted more IL-12p40 in the absence of IL-10 than cells from their wild-type cohorts and translated to a biased pattern of cytokine production in antigen-driven cultures. In this respect, DC matured in the presence of IL-10 have an impaired capacity to drive Th1-type responses caused by their poor synthesis of IL-12 (33). Not only were more CD4+IFN-{gamma}+ cells detected in the sdLN in the absence of IL-10, but the amount of IFN-{gamma} per cell was increased as judged by the intensity of IFN-{gamma} staining.

Although analysis of the immune effector response was not an objective of our study, the data presented here are consistent with the view that, following exposure of C57BL/6 mice to a single dose of RA larvae, IL-12-mediated Th1-type immune effector mechanisms dominate and are responsible for vaccine-induced protection. It is well documented that in the absence of IL-12, protective immune responses are substantially reduced, concurrent with an elevated Th2-type immune environment (10,20), whilst the absence of IL-10 leads to the induction of very high levels of immunity (10). However, in the absence of strong Th1-polarizing cytokines (10, 20), following multiple vaccination (10,43) or in other strains of mice (12), antibody-mediated parasite clearance has an additional protective role (11).

In conclusion, we show that IL-10 plays an important role regulating the very early events of immune stimulation both in the skin and sdLN after exposure to RA larvae. The inhibitory functions of IL-10 appear to operate against accessory cells (e.g. DC), but it remains a challenge for us to establish the cellular origin of IL-10 in the skin. In this respect, IL-10 derived from dermal CD4+ cells is an important factor in the persistence of Leishmania major (44). In the absence of IL-10, IL-12 from skin-derived DC and/or macrophages (4) is enhanced leading to increased MHC II and co-stimulatory molecule expression, and finally resulting in strong Th1 cell polarization. In contrast, the absence of IL-12p40 leads to an abundance of IL-10 which down-regulates MHC II and ultimately affects the efficiency of T cell activity in the sdLN. This study highlights the role of early IL-10 as a brake on IL-12-mediated immune priming and suggests that approaches to control its influence, whilst at the same time limiting inflammation, are important issues to address during the design of anti-parasite vaccines.


    Acknowledgements
 
This work was supported by a Wellcome Trust University Fellowship to A. P. M. (grant 056213). S. K. was supported by a PhD studentship awarded by the Royal Thai Government. We thank George Pitchford and Niall MacDougall at Cookridge Hospital, Leeds, for access to the 60Co source, Ann Bamford for maintaining the parasite life cycle, and Rachel Millard for help with the intracellular cytokine staining.


    Abbreviations
 
APC—antigen-presenting cell

DC—dendritic cell

p.v.—post-vaccination

RA—radiation attenuated

sdLN—skin-draining lymph node

SSP—soluble schistosomular antigen preparation


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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