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. 1999 Feb;96(2):298–306. doi: 10.1046/j.1365-2567.1999.00698.x

The role of urocanic acid in UVB-induced suppression of immunity to Trichinella spiralis infection in the rat

J Garssen *, M Norval *, J Crosby †, P Dortant *, H Van loveren *
PMCID: PMC2326747  PMID: 10233709

Abstract

The naturally occurring trans-isomer of urocanic acid (trans-UCA), found in the stratum corneum, absorbs ultraviolet light (UV) and isomerizes to the cis-form. Cis-UCA has been shown to impair some cellular immune responses, and has been proposed as an initiator of the suppression that follows UV irradiation. UVB exposure leads to an increase in cis-UCA in the skin of rats from about 10% to 40% of the total UCA. Previously it has been demonstrated that UVB lowers immune responses to Trichinella spiralis after oral infection of rats with the parasitic worm. In the present study we investigated the role of cis-UCA in the control of this parasitic infection. Rats were infected orally with T. spiralis and injected with different doses of cis- or trans-UCA subcutaneously. Mitogenic responses and the mixed lymphocyte reaction were not affected by either isomer. In contrast, the number of T. spiralis larvae in muscle tissue of infected rats was increased significantly in the cis-UCA-treated animals compared with the trans-UCA-treated animals. In addition, delayed-type hypersensitivity (DTH) to T. antigen in infected rats was significantly impaired by cis-UCA but not by trans-UCA. If rats were injected with a monoclonal antibody with specificity for cis-UCA 2 hr prior to UVB exposure, the UVB-induced suppression in DTH to T. spiralis and the increase in larvae counts were significantly inhibited compared with rats that were similarly injected with a control antibody. Thus cis-UCA can inhibit the specific resistance to parasitic infections and acts as an important mediator of UVB-induced suppression of immunity to T. spiralis in the rat.

INTRODUCTION

The mode of action by which ultraviolet B light (UVB; 280–315 nm) radiation impairs specific and non-specific immune responses is one of the main topics in photoimmunology. As UVB is not able to penetrate beyond the first few layers of the epidermis, UVB-induced immunosuppression is likely to be initiated by chromophores present in the epidermis. One such photoreceptor is urocanic acid (UCA), located predominantly in the stratum corneum, where it is synthesized as the trans-isomer from histidine by deamination.1,2 It represents about 0·7% of the dry weight of the epidermis and is a major absorber of UV in the skin.3 On exposure, trans-UCA converts to cis-UCA in a dose-dependent fashion until the photostationary state is reached, when approximately equal quantities of the two isomers are present. Once formed, cis-UCA persists at an increased level in the skin for about 2 weeks after termination of UVB radiation before gradually returning to a background low level. Systemically, it can be detected transiently in serum following UVB exposure and for longer periods of time in urine.4,5

UCA was originally proposed to have an important function as a ‘natural’ sunscreen to protect against sunburn,6 and to act as a photoprotecting agent against damage to DNA in the skin.7 More recently it was postulated that cis-UCA may be an initiator of the immunomodulation that follows UVB exposure.1 A variety of in vivo and in vitro experimental systems has provided evidence that cis-UCA can induce immunomodulation.8–11 Analysis of the action spectrum of suppression of contact hypersensitivity (CHS) by UVB radiation and the absorption spectrum of trans-UCA revealed that they were similar.1 In histidase-deficient mice, which have less than 10% of the normal amount of cutaneous UCA, UVB-induced suppression of CHS was limited, while in mice fed a diet rich in histidine, resulting in increased levels of cutaneous UCA, the suppression in CHS induced by UVB was more severe than in normal mice.8 Another example was the effect of treatment of mice with cis-UCA prior to infection with herpes simplex virus (HSV). On challenge with the virus at a later date, the delayed-type hypersensitivity (DTH) response was suppressed and antigen-specific T cells were generated in the spleens of such animals capable of transferring the suppression to other mice.12–14 However, the mechanism of action of cis-UCA is not clear at the present time. Published work indicates that it may act by inducing epidermal cells to release tumour necrosis factor-α (TNF-α),15 through histamine-like receptors16,17 or via c-AMP modulation in skin fibroblasts.18

Many studies monitoring the effects of UVB on immune responses to infectious agents have concentrated on bacterial and viral infections, occurring mainly at cutaneous sites in murine models. There is little information available on parasites, apart from Trichinella spiralis19 and Leishmania, where irradiation suppressed immunity,20,21 and Schistosoma, where it did not.22 In the present study the role of cis-UCA in modulating immune responses in the rat was examined using three approaches. Firstly, the isomerization of UCA in rat skin was assayed following UVB irradiation. Secondly, the effects of treatment of rats with UCA isomers on the morphology of lymphoid tissues were measured, as well as on mitogen and mixed lymphocyte responsiveness of spleen cells. Thirdly, the involvement of cis-UCA in the UVB-induced suppression of the resistance to the nematode T. spiralis was monitored. Following oral infection with T. spiralis larvae, the encysted larvae survive in the acid environment of the stomach, enter the small intestine and become sexually mature within 1–3 days. After copulation, the adult viviparous females penetrate the intestinal mucosa where they produce larvae. These new-born larvae migrate to the striated muscle tissues where they are encysted, completing their life cycle. Expulsion of the adult worms from the gut is mediated by inflammation of the bowel, which becomes evident about 6 days after the primary infection. Additionally, around the cysts (in striated muscle tissues) inflammatory responses occur, which are strictly T-cell dependent as they are virtually absent in congenitally athymic mice or rats. We have shown previously that UVB irradiation of rats infected with T. spiralis leads to the suppression of immune responses to the parasite.19 The role of cis-UCA in this process was assessed by treating infected rats with UCA isomers and subsequently measuring larvae counts in the carcass and monitoring DTH responses to Trichinella antigen. In addition, a monoclonal antibody with specificity for cis-UCA5 was used prior to UVB exposure in rats infected with T. spiralis. This antibody has been demonstrated previously to abrogate some of the effects of UVB on the immune system, such as reversing the suppression of DTH to HSV induced by UVB in mice infected with this virus.23

MATERIALS AND METHODS

Animals

Male outbred WU rats (Wistar–Unilever) (specific pathogen-free; SPF), 6–8 weeks old, were obtained from the breeding colony of RIVM (Bilthoven, the Netherlands). Inbred male PVG rats (SPF), 6–8 weeks old, were obtained from Harlan Sprague–Dawley (Zeist, the Netherlands). The animals were housed separately in macrolon cages and were provided with commercial rat chow (Trouw, Nijkerk, the Netherlands) and tap water ad libitum. Temperature and humidity were kept constant for the exposure time (22° and 50% humidity). Light was provided by a Philips 16, yellow fluorescence lamp, emitting no UVB, and the light/dark schedule was constant, 12/12 hr.

UVB exposure

For UVB exposure, FS40 Westinghouse sunlamps (Westinghouse, Bloomfield, NY) were used. By measuring with the Optronics OL-752-O-PMT spectrophotometer (Philadelphia, PA), the FS40 source emits the total spectrum of UV, of which 0·6% is UVC (250–280 nm), 53% UVB (280–315 nm) and 46% UVA, with an energy load of ≈3·26 W/m2, at 25 cm beneath the two lamps. The peak emission is at 313 nm.

Acute exposure

Rats were shaved on their dorsal sites and subsequently exposed to a single dose of 1500, 3000, 4500 or 6000 J/m2 UVB radiation [one minimum erythemal dose (MED) is about 3000 J/m2 with the FS40 lamps]. The rats were killed 30 min after the exposure. Swiss rolls of the dorsal skin and ears were frozen in liquid nitrogen and stored at −70° until analysis for UCA isomers.

Semi-chronic exposure

Rats were shaved on the back every 3 days and were irradiated for 15 min daily (maximal 6 weeks). Controls were treated similarly but not irradiated. The intensity of the lamps was dimmed, in order to expose the groups to either 25%, 50% or 100% of the maximal intensity of the lamps. Thus each experiment contained four groups of animals (n = 8 or 10), a low dose UVB group (375 J/m2), a middle dose UVB group (750 J/m2), a high dose UVB group (1500 J/m2) and an unirradiated control group. The day after the last exposure the animals were killed and Swiss rolls of the dorsal skin were frozen in liquid nitrogen and stored at −70° until analysis for UCA isomers. In the infection studies the animals were exposed to UVB daily (1500 J/m2; i.e. 0·5 MED per day) for 1 week starting 7 days after oral infection with T. spiralis. Thus in infection studies, the animals were exposed for 7 consecutive days.

Analysis of UCA isomers

After UVB exposure (acute or semi-chronic), a piece of dorsal skin or ear was weighed (20–40 mg) and homogenized as described previously.24 UCA isomers were analysed by high-performance liquid chromatography as outlined previously.24

Treatment with cis-and trans-UCA

The rats (n = 5–8 for each dose) were injected subcutaneously (s.c.) with 50, 100 or 200 μg cis-UCA, prepared as outlined previously,24 or 200 μg trans-UCA (Sigma, Zwÿndrecht, the Netherlands), dissolved in 0·1 ml phosphate-buffered saline (PBS), three times a week for 4 weeks. Control rats (n = 5–8) were injected similarly with 0·1 ml PBS. In infection studies the rats were injected with the compounds starting 1 week prior to infection.

Gross pathology

Animals were observed daily and macroscopic changes of the skin, such as oedema, erythema, discoloration (other than erythema), were recorded. At necropsy, the rats were weighed and the mandibular, axillary, brachial, popliteal lymph nodes, spleen and thymus were also weighed.

Histopathology

Half of each spleen, the thymus, mandibular lymph nodes and skin of the left side of the body were fixed in neutral aqueous phosphate-buffered 4% formaldehyde. The formalin-fixed tissues were embedded in paraplast and 5-μm thick tissue sections were prepared and stained with haematoxylin and eosin. Microscopic examination was performed without knowledge of the treatment. The data were documented with the PATHOS data acquisition system (Pathology Operating Systems Ltd, Harrogate, UK).

For the T. spiralis infection studies, parts of the tongue were fixed in neutral aqueous phosphate-buffered 4% formaldehyde. The formalin-fixed tissues were embedded in paraplast and 5-μm thick sections were prepared and stained with Giemsa for counting larvae present in the tongue. The number of larvae was counted in two sections per animal and expressed as number/cm2. Quantification was carried out using a computerized morphometric image analysing system (IBAS 200, Kontron, Munich, Germany).

Isolation of lymphocytes

Cell suspensions were prepared by gently pressing half of each spleen through a stainless steel screen in a tube with 10 ml medium [Iscove’s medium supplemented with 5% inactivated fetal calf serum (FCS), 100 μg/ml streptomycin and 100 IU/ml penicillin]. The cells were washed (300 g, 10 min, 4°) and filtered through glass-wool, washed once more, and finally resuspended in 10 ml medium (Iscove’s medium supplemented with 10% FCS, 100 μg/ml streptomycin and 100 IU/ml penicillin). Using the Coulter Counter (Counter Electronics Ltd, Mÿdrecht, the Netherlands) the cells were counted, and suspensions at appropriate concentrations were prepared.

Lymphocyte transformation test (LTT)

Three different mitogens were used to stimulate lymphocyte proliferation: concanavalin A (Con A) (Jansen Chimica, the Netherlands), phytohaemagglutinin (PHA; Wellcome, Dartford, UK) and pokeweed mitogen (PWM) (Gibco, Breda, the Netherlands). Splenic lymphocytes were suspended (4·5×105 cells/well) in Iscove’s medium supplemented with 10% FCS, 100 μg/ml streptomycin and 100 IU/ml penicillin. Each well in a 96-well round-bottomed plate was filled with the prepared cell suspension and optimized concentrations of mitogens (3 μg/ml PHA, 3 μg/ml Con A or 30 μg/ml PWM) or medium and incubated at 37° and 5% CO2. After 48 hr of culture, [3H]thymidine (1·0 μCi/well; (Sigma, Bornem, Belgium) was added to each well and the incubation was continued for a further 24 hr. The cells were harvested on glass-fibre filters (LKB-Wallac, Breda, the Netherlands) using a multiple cell culture harvester (LKB-Wallac, Finland). Scintillation liquid (LKB, UK) was added to the filters and the activity was counted in a liquid scintillation counter (LKB-Wallac). The LTT response (in c.p.m.) was expressed as the difference in [3H]thymidine uptake of lymphocytes (cell proliferation) cultured with mitogens and lymphocytes cultured with medium alone. Each test was performed in sixfold replicates. For calculation of the results from each animal, the highest and lowest values were not used.

Mixed lymphocyte reaction (MLR)

Lymphocytes (100 μl; 1×106 WU cells/ml) were co-cultured with 100 μl of stimulator lymphocytes (1×106 PVG spleen cells/ml) in each well. Splenocytes or lymph node cells of outbred WU-rats were used as responders and splenocytes of inbred PVG rats were used as allogeneic stimulators and were γ-irradiated (2000 rads, 15 min) in order to prevent their proliferation. The cells were incubated in Iscove’s medium supplemented with 15% FCS, 100 μg/ml streptomycin, 100 IU/ml penicillin and 0·035% mercaptoethanol (Serva, Heidelberg, Germany). After 96 hr of co-culture, [3H]thymidine (1·0 μCi) was added to each well and the cells were harvested 24 hr later as described above. Responders (WU cells) were also co-cultured with their syngeneic γ-irradiated lymphocytes (obtained from the same animal). The MLR (in c.p.m.) was the difference in [3H]thymidine uptake of lymphocytes cultured with allogeneic cells and lymphocytes cultured with syngeneic cells. For each animal the MLR was performed in sixfold replicates and for calculation of the results from each animal the highest and lowest values were not used.

Infection of rats with T. spiralis and treatment with UCA isomers and cis-UCA monoclonal antibody

Rats were infected orally with 1000 T. spiralis larvae in 0·5 ml PBS, as described elsewhere.19 In some experiments the rats were injected s.c. with UCA isomers (three times a week during 4 weeks, different doses), while in other experiments rats were treated intraperitoneally (i.p.) with a monoclonal antibody to cis-UCA or a control isotype-matched antibody (2 hr prior to each UV exposure), as described previously.23

Quantification of larvae counts

Rats were killed by CO2 asphyxiation 45 days after infection, skinned, and eviscerated. The carcasses were cut into pieces of 0·5 cm3, and 100 g tissue was mixed for 2 hr at 37° with 1 litre of H2O (tap water), 10 ml 36% HCl and 7·5 g pepsin (SVM, Bilthoven, the Netherlands). The suspension was divided into two containers and after 15 min the larvae settled on the bottom of these. In order to remove bones, the larvae were filtered through a stainless steel screen and washed in PBS. For the determination of numbers of larvae, samples were taken from this solution.22 The larvae were stirred in a solution of 400 ml PBS for 5 min and 10 drops, each of 0·05 ml, were placed on a Petri dish. The total number of larvae in these drops was counted with a microscope (40×). This procedure was performed in duplicate. Finally the total number of larvae in the carcass was calculated.22 In addition, larvae counts were performed histopathologically in tongue tissue (see Histopathology).

Preparation of T. spiralis antigen

Trichinella spiralis larvae were homogenized in 5 ml extraction buffer (10 mm Tris–HCl, pH 8·0; 2 mm EDTA; 2 mm phenyl methyl sulphonyl fluoride; 1 μg/ml leupeptin and 1 μg/ml pepstatin) in potter tubes. After centrifugation (1000 g) of the homogenate, the supernatant was stored. New extraction buffer (5 ml) was added, and the same procedure was repeated four times. After the last preparation, the homogenate was added to stored supernatants. This suspension was stirred for 30 min in a rotator. Subsequently, the suspension was centrifuged for 60 min at 50 000 g. The supernatant was used as crude extract Trichinella antigen. Protein content was determined by Lowry’s assay.

DTH to T. spiralis

Seventeen days after infection the animals were challenged s.c. in the ear pinnae with Trichinella antigen (25 μl of 100 μg/ml Trichinella antigen solution). Prior to ear challenge and 24 hr after ear challenge, the ear thickness were measured using an engineer’s micrometer (Mitutoyo Digimatic, Veenendaal, the Netherlands). In each experiment the increase in ear thickness in uninfected animals was measured to give the background response. There were eight animals per group.

Statistics

For the statistical analysis of data from the basal immune function tests, one-way anova was used. Statistical differences between groups were determined using the Student’s t-test based on the anova. For the analysis of histopathological data, a non-parametric test, the Wilcoxon test, was used except for the larvae counts, which were analysed using the Student’s t-test. The data were calculated with Lotus 2.4 (Lotus Development European Corporation, Staines, UK) and transferred to a statistical program called Minitab (Minitab Inc., State College, PA).

RESULTS

Analysis of UCA content in rat skin after UVB irradiation

Analysis of the dorsal skin from experiments in which animals were exposed to different doses of UVB per day for 3–42 days, revealed that the percentage of cis-UCA in dorsal skin of the rats was low. The highest percentage of cis-UCA was demonstrated in rats that were UVB-exposed daily for 7 consecutive days (Fig. 1).

Figure 1.

Figure 1

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Effect of daily UVB exposure for 3–42 consecutive days on the isomerization of trans-UCA to cis-UCA in dorsal skin. The isomerization is shown as percentage cis-UCA of the total amount of UCA (mean±SEM, five rats per group). If no SEM is indicated the SEM is too small to be seen. Lines represent non-irradiated rats (▵) and UVB-irradiated rats (daily dose of 375 J/m2 (♦), 750 J/m2 (•) and 1500 J/m2 (□).

In a second set of experiments rats were irradiated with a single dose of UVB and UCA isomers were measured in dorsal skin and ears. The highest percentage of cis-UCA was found in the ears and reached ≈40% of the total amount of UCA (Fig. 2). The concentration of total UCA was not affected by UVB exposure in the ears (360 ng/mg wet weight skin) or the dorsal skin (150 ng/mg wet weight skin).

Figure 2.

Figure 2

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Effect of a single dose of UVB radiation on the isomerization of UCA in the dorsal skin (•) and ears (○). Isomerization is presented as percentage cis-UCA of the total amount of UCA (30 min after exposure). Data are presented as mean±SEM (five rats per group). If no SEM is indicated the SEM is too small to be seen.

The effect of subcutaneous cis-UCA on the immune system

Analysis of the body weight and the weight of lymphoid organs in animals that were injected s.c. with 12 doses of cis-UCA ranging from 50 to 200 μg did not demonstrate any significant changes from the trans-UCA or control groups (data not shown). There was a tendency to increased cortical apoptosis in the thymus of the cis-UCA-injected rats in comparison with the control group. In the mandibular lymph nodes a decrease in the number of secondary follicles in the germinal centres in the two highest doses of cis-UCA groups was present (Fig. 3).

Figure 3.

Figure 3

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Micrographs of thymus and mandibular lymph node showing representative changes in cis-UCA-treated rats (rats were injected with the UCA isomers during 4 weeks, three injections per week, and killed 1 day after the last injection). (a) Thymus of a control rat (received vehicle only). me=medulla. H&E. ×83. (b) Thymus of a rat treated with 12 doses of 200 μg cis-UCA; an increase in cortical apoptosis, especially in the presumed epithelial free areas (indicated with arrows) is observed. me=medulla. H&E. ×83. (c) Mandibular lymph node of a control rat that received vehicle only; a marked development of germinal centres (indicated with arrows) is present. H&E. ×33. (d) Mandibular lymph node of a rat treated with 12 doses of 200 μg cis-UCA; development of secondary follicles is limited. H&E. ×33.

Proliferation of splenocytes, induced by different mitogens (Con A, PHA and PWM), was not affected by cis- and trans-UCA (Fig. 4a). Neither cis- nor trans-UCA affected the net MLR response of splenocytes (Fig. 4b).

Figure 4.

Figure 4

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Effects of 12 subcutaneous doses of cis- and trans-UCA (three times a week during 4 weeks; 28 days protocol) on the mitogen response (LTT) (a) and the mixed lymphocyte reaction (MLR) (b) of lymphoid cells from the WU rat in response to co-culture with allogeneic PVG spleen cells or lymph node cells. LTT and MLR are presented as net c.p.m. responses (the control responses are subtracted). Bars represent the mean±SEM of six rats per group (0, 50, 100, 200 μg cis-UCA) (□ = lowest dose up to ▪=highest dose cis-UCA from left to right in each set of five bars). The furthest right bar of each set of five bars represents, as a control, 200 μg trans-UCA (░).

The effect of subcutaneous cis-UCA on T. spiralis larvae numbers and DTH to Trichinella antigen

In order to analyse whether UCA can affect immunity to T. spiralis, rats were infected orally 1 week after the start of the injections of UCA isomers. The larvae counts in muscle tissue (carcasses, Fig. 5a, and tongue tissue, Fig. 5b) were significantly increased if the animals were treated with cis-UCA. Trans-UCA did not induce any significant effect on the clearance of the parasite (Fig. 5).

Figure 5.

Figure 5

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Effects of 12 subcutaneous doses of cis-and trans-UCA (three times a week during 4 weeks) or PBS on larvae counts in (a) carcasses and (b) tongue tissue in rats that were infected with Trichinella spiralis (orally) 1 week after the start of the injections. Each group consisted of 8 rats. Data are presented as means±SEM (*P < 0·05 compared with control group). **P < 0·05 compared with 50 μg cis-UCA.

In the carcasses, 100 μg cis-UCA increased the number of parasites whereas 50 μg cis-UCA did not induce any significant effect (Fig. 5a). The number of larvae in tongue tissue from cis-UCA-treated animals was significantly increased (P < 0·05) if the animals were treated with 50 or 100 μg cis-UCA. The increment in number of larvae in the 50 μg cis-UCA group was significant compared to the trans-UCA (50 μg)-treated group. The number of larvae in tongue tissue in both trans-UCA-treated groups was not significantly different from the control (PBS) group (Fig. 5b).

DTH was dose-dependently suppressed by cis-UCA, whereas trans-UCA had no effect at all. In the group that was injected s.c. with 100 μg cis-UCA daily, no significant DTH response could be detected (Fig. 6). DTH swelling reactions in cis-UCA-treated animals (both doses) were significantly impaired compared to trans- UCA-injected animals (in both cases, 50 and 100 μg, P<0·05).

Figure 6.

Figure 6

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Effects of 12 subcutaneous doses of cis- and trans-UCA or PBS on DTH to Trichinella antigen in rats that were infected orally, or not infected, 1 week after the start of the PBS or UCA isomer injections. Hatched (even) bars represent Trichinella-infected rats and open (odd) bars represent non-infected rats (background swelling response). Each group consisted of eight rats. Data are presented as means±SEM. P-values in the figure represent significance levels of ear swelling responses compared with background ear swelling measured in non-infected animals (NS=not statistically significant). DTH (ear swelling) responses in cis-UCA-treated rats were significantly impaired compared with trans-UCA-injected rats (for both 50 and 100 μg cis-UCA P < 0·05 compared with respectively, 50 and 100 μg trans-UCA).

The effect of a monoclonal antibody to cis-UCA on the UVB-induced increase in larvae and suppressed DTH

If animals infected with T. spiralis were injected with a monoclonal antibody with specificity for cis-UCA prior to each UVB exposure, the increment in larvae numbers due to UVB exposure was significantly inhibited (Fig. 7). In animals treated with the control antibody no significant inhibition of the UVB effect was detected. This was also true for the effect of UVB on the DTH to Trichinella antigen (Fig. 8).

Figure 7.

Figure 7

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Effect of i.p. injections with a monoclonal antibody to cis-UCA or a control antibody prior to each UVB exposure on larvae counts in tongue tissue of T. spiralis-infected rats. The rats were infected orally 1 week prior to the first UVB exposure. Each group consisted of eight rats. Data are presented as mean±SEM.

Figure 8.

Figure 8

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Effect of i.p. injections with a monoclonal antibody to cis-UCA or a control antibody prior to each UVB exposure on DTH to Trichinella antigen in rats that were infected 1 week (orally) prior to the first UVB exposure. Swelling responses in non-infected rats were subtracted. Each group consisted of eight rats. Data are presented as mean±SEM.

DISCUSSION

While most work on UVB-induced immunomodulation in experimental systems has concentrated on contact hypersensitivity and carcinogenesis, several studies have indicated that a suppressed resistance to a variety of infectious agents results following UV exposure. This effect is not confined to skin-associated infections and has been shown, for example, in Mycobacterium infections in the mouse25 and Listeria monocytogenes26 or T. spiralis infections in the rat.19,27 It is well known that UCA isomerization is involved in UVB-induced immunomodulation.1,2,5,9,10 Whether cis-UCA can impair resistance to T. spiralis infections in the rat and whether the UCA isomerization is involved in initiating the UV-induced immunosuppression to T. spiralis in the rat were investigated in the present study.

Initial experiments showed that the skin of rats contained approximately the same quantity of UCA as mouse skin (rat dorsal skin 150 ng/mg and ears 360 ng/mg wet weight; C3H/HeN mice dorsal skin 160 and ears 370 ng/mg wet weight).24 In both dorsal and ear skin of rats, UV exposure led to trans to cis isomerization, but the percentage of cis-UCA was higher in the ear compared with the dorsal skin. Although the animals were shaved prior to irradiation, the dorsal skin is full of hair follicles that may have protected against effective UCA photoisomerization to some extent. In contrast, the ears of rats are relatively devoid of hair. In the ears the percentage of cis-UCA found after irradiation was approximately the same as in the ears of haired C3H/HeN mice24 or hairless Skh-1 mice28 receiving the same exposure in terms of MED from a similar broadband UVB source. The percentage cis-UCA in the skin was maximal after 1 week of chronic UV exposure. After longer periods of UV exposure the increment in percentage of cis-UCA was not present in the skin, which may have been due to epidermal thickening. Systemically increased percentages of cis-UCA are detectable in urine and serum of human subjects for longer periods than 7 days.4 For this reason the peak in percentage cutaneous cis-UCA at day 7 after the start of the UV exposure does not mean that it necessarily peaked in other sites at the same time.

The immunotoxic activity of UCA isomers was analysed using an approach that was adapted from the guidelines for toxicity testing after oral exposure to chemicals; however, no doses were tested that induced toxic effects such as body weight changes and/or changes in organ weights.29 In previous studies it was demonstrated that the ears of mice contain ≈8 μg UCA per cm2. After one dose of 1 MED UVB, one-third is found as cis-UCA. The rats were shaven prior to UVB exposure and thus ≈60 cm2 shaven skin was exposed to UVB (total-body exposure). This means that the UCA doses that were injected were probably not very different from photoreceptor concentrations in animals that were exposed to UVB for, for example, 7 consecutive days.

The 28-day testing protocol did not lead to any significant effect on the MLR and mitogenic responsiveness of lymphoid cells from cis-UCA or trans-UCA-treated rats. Additionally, no significant differences were found in the weights of lymphoid tissues (thymus, spleen and lymph nodes), although there were some pathological effects in the lymph nodes and thymi of animals treated with cis-UCA (i.e. cortical apoptosis).

Cis-UCA treatment led to an increase in the total number of larvae per animal or per mm2 tongue tissue after oral infection with T. spiralis, indicating a reduction in the resistance mechanism of the host. In these experiments the animals were injected with UCA isomers using the 28-day testing protocol, and were infected after three injections (i.e. 1 week after the start of the injection protocol). One specific cellular immune response, the DTH to T. spiralis, was significantly suppressed by cis-UCA. The degree of cis-UCA-induced immunosuppression was dependent on the amount of cis-UCA administered. The lymphoproliferative response in vitro to Trichinella was not tested, although it was shown previously to be suppressed by UVB exposure in infected rats, while the response to Con A was not affected.27

The protocols for the cis-UCA and the UVB exposure studies in the T. spiralis rat model were not identical due to technical and guideline reasons. The cis-UCA was administered over a period of 4 weeks, starting 7 days before giving the parasite. Thus modulations in immune responses might have occurred prior to infection. In contrast, UVB exposure was most effective if the rats were irradiated during the second week after oral infection. In order to validate the role of cis-UCA in UVB-induced suppression of immunity to T. spiralis, further experiments were performed using a monoclonal antibody with specificity for cis-UCA.5 This antibody has been shown previously to reverse the suppression in DTH caused by UVB irradiation in a mouse model of HSV infection.23 Rats were injected with the antibody prior to each UVB exposure. This resulted in a partial restoration of the suppressed DTH to Trichinella and to a partial reversal of the increased larvae counts in muscle tissue. Thus, although the antibody did not completely abrogate the effects of UVB exposure on these two parameters, it did have considerable activity. It could be that sufficient antibody was not administered on each occasion to stop the UVB downregulation entirely, or that other mediators in addition to cis-UCA are involved. However, if these antibody results are considered together with the cis-UCA results discussed above, evidence is presented to demonstrate an important role for cis-UCA in initiating the effects of UVB irradiation on T. spiralis infection in the rat.

The rats were inoculated orally with the parasite, with subsequent spread of larvae from the gut to muscle tissue. The antigen-presenting cells that are involved in initiating the immunological control of the intestinal parasite are therefore very different from those that are present in the skin and that can be affected directly by UV exposure (e.g. Langerhans’ cells). This implies that cis-UCA, once formed by photoisomerization in the epidermis, can either initiate a cascade of responses starting in the skin and then having systemic effects or, as it can be detected in blood and urine following irradiation, it could be immunomodulatory in sites other than the skin. Evidence has been published from CHS models in mice indicating that cis-UCA may act by inducing the synthesis of TNF-α from keratinocytes or Langerhans’ cells,30,31 but other studies have not confirmed this suggestion.32,33 There is also the possibility that histamine and prostaglandins may be involved.34 Recently the UVB-induced suppression of DTH to alloantigen was shown to be partially reversed by antibodies to interleukin-10 (IL-10) administered before irradiation35,36 and it will be particularly interesting to examine the role of IL-10 in the lowered resistance to T. spiralis induced by cis-UCA.

In conclusion, the present study indicates that photoisomerization of UCA is involved in the immunosuppressive effects of UVB exposure, leading to decreased immunity to a systemic parasitic infection.

Acknowledgments

The work was supported by Grants EV5 V-CT91-0028 and ENV4-CT96-0912 of the European Community ‘Environmental Programme’. We thank Professor Dr J.G. Vos for critically reviewing the manuscript.

References

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