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Schistosoma mansoni, S. haematobium, and S. japonicum: early events associated with penetration and migration of schistosomula through human skin
Experimental Parasitology 102 (2002) 99–108 www.elsevier.com/locate/yexpr
Schistosoma mansoni, S. haematobium, and S. japonicum: early events associated with penetration and migration of schistosomula through human skin Yi-Xun He, Lin Chen, and K. Ramaswamy* Department of Biomedical Sciences, College of Medicine, University of Illinois, Rockford, IL 61107, USA Received 12 February 2002; received in revised form 9 October 2002; accepted 18 February 2003
Abstract Migratory pattern of schistosomula of Schistosoma mansoni, S. haematobium, and S. japonicum through human skin were analyzed in skin organ cultures. These studies showed that the schistosomula of S. mansoni and S. haematobium has similar migratory patterns through human skin. During the first 24 h after infection nearly 90% of S. mansoni and S. haematobium schistosomula were present only in the epidermis. Majority of the schistosomula were found in the dermis only after 48 h and they appear to reach the dermal vessels around 72 h after infection. Migratory pattern of S. japonicum on the other hand was significantly different from the other two species in that over 90% of the parasites had already reached the dermis within the first 24 h and schistosomula were present in the dermal vessels within 2 h after infection. Analysis of the cytokine pattern at 8 h after infection by a macro gene array and RT-PCR analysis showed that out of 24 different cytokines analyzed only IL-1ra, IL-10, and TNF-a were increased in the human skin following infections with S. mansoni and S. haematobium, whereas, after infection with S. japonicum there was significant increases in IL-1b, IL-1ra, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a. Immunohistochemical analysis of epidermal sheets showed focal accumulation of HLA-DRþ cells in areas where schistosomula of S. mansoni had entered the human skin. Ó 2003 Elsevier Science (USA). All rights reserved. Index Descriptors and Abbreviations: Trematode, S. mansoni, S. haematobium, S. japonicum, human skin, schistosomula, migration, cytokines, HLADRþ cells. Interleukin (IL), tumor necrosis factor (TNF), intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
1. Introduction Human schistosomiasis is a water-borne infection caused mainly by three different species of schistosomes, Schistosoma mansoni, S. japonicum, and S. haematobium. Current estimates show that more than 200 million people are infected with these parasites and another 300 million are at risk (WHO, 1993). Human infections occur when cercariae, the infective stages of these parasites, penetrate intact skin and enter into the body (Wheater and Wilson, 1979). This is the only known route of entry for this parasite into human. Yet unfor-
*
Corresponding author. Fax: 1-815-395-5666. E-mail address: [email protected] (K. Ramaswamy).
tunately, very little has been studied on the mechanism of penetration or host response to these parasites in the human skin (Bartlett et al., 2000; Fusco et al., 1993; Khammo et al., 2002; Kusel, 1970) and no information is available on the kinetics of schistosome migration through human skin. To begin to understand the early host responses and migratory pattern of various schistosomula through human skin, we have used a human skin organ culture system (Ramaswamy, 1998) that allowed us to infect human skin pieces ex vivo with cercariae of all three schistosomes and monitor kinetics of schistosomula migration. Once thought to be primarily a target for immune attack during various reactions, the skin, particularly the epidermis, has been shown to actively participate in the generation of immunologic responses affecting the
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00024-9
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skin and the entire immune system (Katz, 1993). Advances in immunodermatology during the past one decade has shown that epidermal cells, specifically keratinocytes, Langerhans cells, and epidermal T lymphocytes can elaborate an array of cytokines into the microenvironment that form a meaningful network of immune responses in the epidermis which is important in the initiation of an appropriate immune responses against invading foreign agents (Uchi et al., 2000). Few studies that have looked to date on the cytokine responses in the mouse skin suggested that the migrating schistosomula are capable of modulating host immune responses in the skin (Angeli et al., 2001b; Ramaswamy et al., 2000; Ramaswamy et al., 1995). At present we do not know whether similar changes are induced in the human skin by all three species of schistosomes. Similarly, we know only very little on the role of epidermal and dermal cells on the host-parasite relationship in human schistosomiasis. In addition to analyzing the migratory pattern of the three schistosomes through skin, the organ culture system has allowed us to compare some of the early cytokine responses associated with parasite entry and migration through the human skin.
2.2. Parasite Biomphalaria glabrata snails infected with S. mansoni and Bulinus truncatus snails infected with S. haematobium were obtained from Dr. Fred Lewis, Biomedical Research Institute, Rockville, MD. S. japonicum life cycle is maintained at our facility in Oncomelania hupensis nosophora snails and ICR strain of mice. Cercariae were collected from the infected snails as described previously (He, 1993; Ramaswamy et al., 1996). In all our studies infections were achieved within 1 h of shedding the cercariae with all three species. 2.3. Histology
2. Materials and methods
Standard paraffin embedded histological procedures were followed for analysis of human skin pieces. Migratory behavior of schistosomula in the human skin in organ cultures was evaluated by staining 6–7 lm serial sections of the skin pieces with hematoxylin and eosin. Number of parasites present in 150–230 consecutive serial sections (which accounted for 1 to 1.5 mm thickness) from each skin piece were counted and the total number of schistosomula present in the epidermis and dermis were determined. Care was taken not to count the same parasite in adjacent sections. Values are presented as percent recovery in epidermis and dermis.
2.1. Human skin organ culture
2.4. Immunohistochemistry
Fresh human foreskin samples collected from SwedishAmerican Hospital, Rockford, USA, was used in these experiments. The use of human skin in these studies were approved by the Institutional Review Board of the College of Medicine at Rockford, University of Illinois. Organ cultures were prepared as described previously (Ramaswamy, 1998). Briefly, fresh foreskin samples cut approximately into 1.5 cm3 cubes were kept in air/liquid biphasic cultures containing RPMI 1640, 5% FBS, 5% human AB serum or 10% fetal bovine serum in some studies, 10 lg/ml gentamicin, and 25 lg/ml fungizone (Gibco, Grand Island, NY) at 37 °C and 5% CO2 in air. Infection was achieved by placing 150–300 cercariae of S. mansoni, S. haematobium, or S. japonicum suspended in 50–100 ll of sterile water under a cover glass directly on to the epidermal surface of the skin and incubated for 60 min at 37 °C. Skin pieces were usually infected within 1–4 h after collection. Following exposures to the parasites the skin pieces were briefly rinsed in RPMI media and incubated further for varying periods of time (1–72 h) in fresh media. Skin pieces from the same individual cultured simultaneously, but not infected with schistosome cercariae, were used as negative controls. Samples for histology were all fixed in buffered formalin. Skin samples were also snap frozen in liquid nitrogen for RT-PCR analysis.
Distribution of Class II major histocompatibility antigen expressing cells were examined in the epidermal sheets of 15 different individuals 48 h after exposure to infection with S. mansoni. Epidermal sheets were prepared by incubating skin pieces in 0.5 M ammonium thiocyanate (Sigma, St. Louis, MO) for 30 min at 37 °C. This allowed the epidermal layer to be peeled off easily. Uninfected skin pieces from the same individual were used as negative controls. After removing the epidermal sheet they were divided into two and mounted on to 2% aminopropyltriethoxysilane (Sigma) coated slides with dermal side up. One of these pieces remained as negative staining control. Epidermal sheets were then washed three times with phosphate-buffered saline (pH 7.3), fixed in acetone for 10 min at room temperature, air dried, and stored at )70 °C until used. Before immunohistochemical staining, slides were thawed to room temperature and epidermal sheets were incubated in a peroxidase blocking solution (Pierce chemicals, Rockford, IL) for 10 min at room temperature. Five percent normal rat serum was used to block non-specific binding sites. To identify Class II positive cells, epidermal sheets were incubated with a mouse anti-human HLA-DR monoclonal antibody (IgG2a , Pharmingen, San Diego, CA) for 1 h at room temperature. Horseradish peroxidase labeled rat anti mouse
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IgG2a antibodies (Pharmingen) were used as the secondary antibody. Color was developed using a metal enhanced diaminobenzidine substrate (Pierce Chemicals) and the epidermal sheets were counter stained with MayerÕs hematoxylin (Fisher Chemicals, Pittsburgh, PA) to identify HLA-DR positive cells. Control samples treated similarly were incubated with a non-specific mouse IgG2a antibody (Pharmingen). 2.5. Cytokine analysis Secreted levels of cytokines for IL-1a, IL-1b, IL1ra, IL-2, IL-10, IL-12, and IFN-c were evaluated in the human epidermal sheets 48 h after exposure to infections with S. mansoni. Briefly, epidermal sheets were peeled off as detailed above, homogenized in a sonicator (Fisher Scientific) in phosphate-buffered saline (pH 7.0) over ice, and centrifuged at 10,000 rpm to collect the soluble proteins in the homogenate. Total protein concentration in the supernatant was estimated using a protein assay kit (BCA kit, Pierce Chemicals). All protein samples were normalized to the same concentration by diluting with PBS. Level of protein in the skin samples varied from 1.5 mg to 2.7 mg/ml. Levels of cytokine in the epidermal cell homogenate was determined using an ELISA using kits purchased from R&D Systems (Minneapolis, MN). Uninfected skin samples from the same individual cultured similarly were used as controls. Ten to 15 foreskin samples were used per group in each experiment. 2.6. RT-PCR analysis of cytokine for IL-1b, IL-1ra, and IL-10 in human skin Human foreskin samples were cultured with 150 cercariae of S. mansoni, S. haematobium, or S. japonicum in RPMI-1640 medium with 10% FBS or in media alone for 4 and 8 h at 37 °C with 5% CO2 . At each time point after culture, skin samples were collected and total RNA extracted with TRIzol reagent (Life Technologies, Rockvill, MD) for reverse transcription using RETROscript (Ambion, Austin, TX). The cDNA of b-actin in each sample was first PCR amplified (Perkin–Elmer, Norwalk, CT) using b-actin-specific primers (Ambion). Based on the band density of b-actin, each sample was then adjusted to approximately the same level. Individual samples were then PCR amplified for IL-1b, IL-1ra, and IL-10, using gene-specific primers for IL-1b (Ambion), IL10 (Ambion), and IL-1ra (Chan et al., 1992). Primers for b-actin, IL-1b, IL-1ra, and IL-10 amplify 294-, 238-, 492-, and 297-bp target fragments, respectively. PCRs were performed as follows: for b-actin, 3 min at 94 °C, 30 s at 55 °C, and 30 s at 72 °C for 30 cycles; for IL-1b and IL-10, 3 min at 94 °C, 30 s at 57 °C, and 30 s at 72 °C for 30 cycles; for IL-1ra, 3 min at 94 °C, 30 s at 60 °C, and 30 s at 72 °C for 30 cycles. The final
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elongation was followed by 5 min at 72 °C for all target cDNA amplifications. The products were resolved on a 1.5% agarose gel and stained with ethidium bromide. Photographs of gels were scanned and band densities were analyzed using NIH Image software. Results are expressed as target band intensity divided by b-actin band density. 2.7. Macro gene array analysis of cytokines in human skin after exposure to infection with schistosomes A macro gene array was performed using a NonradGEArray kit (Catalog # hGEA9912090) purchased from Superarray (Bethesda, MD) to detect cytokine mRNA expression in human skin 8 h after exposure to 150 cercariae of S. mansoni, S. haematobium, or S. japonicum for 8 h. The assay was performed according to the instructions provided by the manufacturer. Cytokine genes analyzed by this gene array included IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL11, IL-12 (p35), IL-12 (p40), IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN-c, TNF-a, and TNF-b. b-Actin and GAPDH were used as controls. Briefly, total RNA extracted with TRIzol was used as a template for synthesis of biotinylated probes using reverse transcription. Membranes spotted with specific cytokine cDNA fragments were then hybridized with these labeled probes. Signal was developed after incubating the membranes with alkaline phosphatase conjugated streptavidin and alkaline phosphatase chemiluminescence substrate. Signal density in each spot was then scanned and analyzed using an NIH Image software. Results are expressed as target band intensity divided by b-actin band density. Experiments were repeated twice using five skin samples per group. 2.8. Statistical analysis Statistical significance of the means of values was tested by one-way analysis of variance using Sigmastat program (Jandel Scientific, San Rafael, CA). All pairwise multiple comparison was done using Student– Newman–Keuls method. Probability value of 1% or lower was considered significant. All values are expressed as means SD.
3. Results 3.1. Migratory pattern of schistosomula through human skin Histological analysis of serial section of human skin samples exposed to 150 cercariae of S. mansoni showed migrating parasites in various layers of the skin at different intervals after exposure to infection. Fig. 1 shows
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Fig. 1. Histological analysis of the migration of schistosomula of S. mansoni (A–G), S. haematobium (I), and S. japonicum (J–L) through human skin in organ cultures. Parasite migration was traced by analyzing 6–7 lm serial sections of the skin pieces. At 4 h after exposure to infection (A), transformed schistosomula of S. mansoni were seen in the outermost layers of the epidermis. By 8 h (B) they had already penetrated deeper into the epidermis and lay horizontal to the basement membrane. Sections examined at 12 h (C) and 16 h (D) after infection showed schistosomula of S. mansoni in the process of penetrating through the basement membrane. By 24 h (E) some of the parasites have already entered the dermis. The schistosomula then migrated further deeper into the dermis by 48 h (F) and reached close to the blood vessels by 72 h (G). Schistosomula of S. japonicum were seen in the dermal vessels (J–L) within 2 h after exposure to infection. However, it took more than 8 h for schistosomula of S. haematobium to cross the epidermal basement membrane (I). Migratory pattern of the schistosomula of S. haematobium through human skin closely resembled those of S. mansoni. Section of uninfected skin (H) prepared at 72 h after culture remained as control. All sections were stained with H&E. Original magnification was X100. Arrows (!) indicate cut sections of schistosomulum. Sections shown are from one of 10 similarly infected human skin pieces.
sequence of events in the migration of schistosomula of S. mansoni, S. japonicum, and S. haematobium through the human skin. During the initial 4 h after exposure to S. mansoni infection, transformed schistosomula were seen in the cuticle and outermost layers of the epidermis
(Fig. 1A). At 8 h after exposure to infection (Fig. 1B) 94% of the schistosomula of S. mansoni were present in the epidermis. Interestingly most of the parasites lay horizontal to the basement membrane before entering into the dermis. Skin sections examined at 12 h (Fig. 1C)
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and 16 h (Fig. 1D) after exposure to S. mansoni infection showed parasites in the process of penetrating through the basement membrane into the dermis. By 24 h (Fig. 1E) only 11% of the parasites had entered the dermis, still 89% were in the epidermis. However, once in the dermis schistosomula of S. mansoni appeared to move faster and deeper (Fig. 1F) until they reached closer to the blood vessels. Most of the parasites observed at 72 h after infection were either inside or closer to the blood vessels in the hypodermis (Fig. 1G). Migratory pattern of S. haematobium schistosomula through human skin appeared to be similar to those of S. mansoni schistosomula. Over 93% of schistosomula of S. haematobium were seen only in the epidermis up to 8 h after exposure to infection (Fig. 1-I). Similar to S. mansoni, the schistosomula of S. haematobium also appear to enter the dermis in large numbers around 48 h and reach closer to the dermal vessels around 72 h after exposure to the infection. Compared to S. mansoni and S. haematobium, schistosomula of S. japonicum appeared to migrate faster through human skin. At 8 h after exposure to infection 73% of the parasite had already migrated to the dermis and by 24 h over 90% of the parasites had reached dermis. Migrating schistosomula of S. japonicum were seen inside dermal blood vessels as early as 2 h after exposure to the infection (Fig. 1J–L). 3.2. HLA-DR expression in human skin after exposure to infection with S. mansoni Immunohistochemical staining of normal human skin showed that HLA-DRþ cells are randomly distributed in the epidermis of control skin (Fig. 2A). However, 48 h after exposure to infection with S. mansoni, there was an increased focal accumulation of HLA-DRþ cells in ar-
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eas where schistosomula has entered into the epidermis (Fig. 2B). 3.3. Levels of cytokine protein in human epidermal sheets after exposure to infection with S. mansoni Previous studies using a human keratinocyte cell line showed significant increases in the expression of IL-1ra when ES products from schistosomula of S. mansoni were added to the cultures (Ramaswamy et al., 1995). Studies in mice showed that in addition to IL-1ra there was also an increase in IL-10 in the skin following infection with S. mansoni (Ramaswamy et al., 2000). Analysis of human skin in organ culture showed a similar increase in IL-1ra and IL-10 within 48 h after exposure to infection with S. mansoni (Fig. 3). Levels of IL-1a, IL-1b, IL-2, IL-12, and
Fig. 3. Analysis of different cytokine levels in human epidermal homogenate 48 h after infection with S. mansoni showed significant increases in the levels of IL-1ra and IL-10 but not in IL-1a, IL-1b, IL-2, IL-12, or IFN-c. Epidermal sheets were prepared as described in Section 2 and homogenized. After normalizing the samples to similar protein concentration, the levels of cytokine in the epidermal cell homogenate were determined by an ELISA using respective kits for each cytokine. Data represent one of three similar experiments using skin pieces from 10 individuals. Control skin samples used were from the same individual. *Significantly different from controls (p < 0:01).
Fig. 2. Distribution of HLA-DRþ cells in human epidermal sheets after infection with S. mansoni (B). Samples of human skin were exposed to 150– 300 cercariae of S. mansoni ex vivo in organ cultures. At 48 h after infection, epidermal sheets were peeled off, fixed in acetone and incubated with mouse anti-human HLA-DR monoclonal antibody (IgG2a ) for 1 h at room temperature. HRP-labeled rat anti-mouse IgG2a and DAB substrate were used to identify positive cells. MayerÕs hematoxylin was used as counterstain. Figures shown are representative of one of 15 different epidermal sheets analyzed. Uninfected epidermal sheet samples collected from the same individual remained as controls (A). Original magnification was 100.
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Fig. 5. Cytokine gene-expression pattern in human skin after infection with S. mansoni, S. haematobium, or S. japonicum. A macro gene array analysis was performed for 23 different cytokines in the human skin 8 h after infection with the three schistosomes in organ culture. After developing the signals, density of each spot was analyzed using an NIH image software and results are expressed as target band density divided by b-actin band density for each sample. Only cytokines that showed significant differences from controls are presented in the graph. These experiments were repeated two times using skin samples from five individuals with similar results. Data presented are from one of these experiments. * and ** significantly different, p < 0:05 and p < 0:01 respectively compared with control. Fig. 4. Analysis of cytokine transcripts for IL-1b, IL-1ra, and IL-10 in human foreskin samples exposed to 150 cercariae of S. mansoni, S. haematobium, or S. japonicum. Total RNA was extracted from skin samples collected at 4 or 8 h after infection and reverse transcribed. Individual samples were then PCR amplified using gene-specific primers. The PCR product were resolved on a 1.5% agarose gel and stained with ethidium bromide (A). Images were then scanned and ratio of target density to b-actin was calculated (B). Data represent one of three similar experiment using skin samples from five to seven individuals per group. *Significantly different from controls ðp < 0:01Þ.
IFN-c did not show any significant difference from control skin samples at this time point (Fig. 3). 3.4. Message levels for IL-1ra and IL-10 in human skin after exposure to infection with S. mansoni, S. haematobium, or S. japonicum We then confirmed the above observation by measuring levels of cytokine mRNA in the skin organ cultures after exposure to infection with S. mansoni. These studies showed that message levels for IL-1ra and IL-10 is significantly increased in the skin by 8 h after exposure to infection with S. mansoni (Fig. 4A). A comparison of the cytokine responses in the skin after exposure to infection with the other two species of schistosomes showed that a similar increase in IL-1ra and IL-10 occurred in the human skin after infections with S. japonicum and S. haematobium (Fig. 4A). IL-1b transcript levels did not show any significant difference from control skin samples (Fig. 4B). Substantially higher levels of IL-1ra transcripts were found in the human skin after exposure to infection with S. japonicum than S. mansoni or S. haematobium. Similarly, compared to 4 h the skin samples collected at 8 h after exposure to infection with all three schistosomes had higher levels of IL-1ra and IL-10 transcripts (Fig. 4B).
3.5. Macro gene array analysis of human skin for cytokine expression after exposure to infection with S. mansoni, S. haematobium, or S. japonicum Given that there are significant differences in the levels of IL-1ra and IL-10 in the human skin in organ culture after infection with all three schistosomes, we then wanted to analyze whether there is any significant difference in the expression of other cytokines as well. We used a macro gene array to analyze message levels of 23 different cytokines (Fig. 5). These studies showed that 8 h after exposure to infection with S. mansoni there was a significant decrease in IL-1a, whereas, IL-10 and TNF-a showed a significant increase. A similar decrease in IL-1a was also evident after exposure to infection with S. japonicum. However, following S. japonicum infection there was a significant increase in the message levels for IL-1b, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a in the human skin. Pattern of cytokine message in the human skin after exposure to infection with S. haematobium was apparently similar to those after S. mansoni infection. However, IL-1a levels did not show any significant changes after exposure to infection with S. haematobium. Compared to control skin there was no significant change in IL-3, IL-4, IL-5, IL-7, IL-9, IL-11, IL-12, IL-13, IL-14, IL-16, IL-17, IFN-c, and TNF-b in the infected skin pieces (data not shown).
4. Discussion Schistosomiasis is still a major human health hazard in many tropical and subtropical areas of the world (WHO, 1993). Human infections occur when cercariae, the infective stages of the parasite, penetrate intact skin and
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enter into the body. Thus, skin appears to be the primary organ of initial contact between the parasite and the human host. Traditionally a mouse model is used to study schistosome–host interaction. Using this model substantial progress has been made during the past four decades in unraveling the mechanism of skin penetration and immunobiology of schistosomiasis in the murine skin (Gui et al., 1995; He, 1993; Ramaswamy et al., 1997; Salafsky et al., 1984). Yet there remains a gap in our understanding of the host–parasite relationship of the three schistosomes in the human skin. In the present study using a human skin organ culture system we have compared the migratory pattern of the schistosomula of S. mansoni, S. haematobium, and S. japonicum, evaluated changes in the cytokine pattern following infection and examined alterations in the distribution of specific cells in the skin as a result of infection. Analysis of the migratory pattern of schistosomula showed that all three species of schistosomes were capable of penetrating human skin in organ culture and were able to migrate through various layers of the skin in a predictable manner. Proteolytic enzymes present in the acetabular glands are believed to help in this process of skin penetration and entry (Dresden et al., 1977; McKerrow et al., 1985; Salter et al., 2000; Stirewalt, 1982). It is often thought that schistosomula find their way into the skin mainly through hair follicles (Stirewalt, 1982). However, our studies suggest that schistosomula of all three species can penetrate and enter into human skin through any skin surface irrespective of hair follicle areas as has been reported in gerbils (BayssadeDufour et al., 1994) and rhesus monkeys (He et al., 1992). Although entry of all three species of schistosomula into human epidermis occurs within minutes, there appears to be significant differences in their migratory pattern through the skin. Schistosomula of S. mansoni and S. haematobium showed apparently similar migratory pattern through human skin. After entering into the skin majority of the schistosomula of S. mansoni and S. haematobium remains in the epidermis for about 24 h before traversing the basement membrane. The reason for such a long stay in the epidermis is not clear. However, it has been postulated that during this time schistosomula may be acquiring host molecules on their surface (Dessein et al., 1981; McLaren et al., 1975; Ruppel et al., 1984; Sher and Benno, 1982) or are developing strategy (Hawn and Strand, 1993; Mei et al., 1996) that potentially allow them to evade host responses (McKerrow, 1997; Pearce and Sher, 1987). Once after crossing the basement membrane they appeared to migrate faster aiming toward the dermal blood vessels. After entering the skin it takes nearly three days for the schistosomula of S. mansoni and S. haematobium to reach dermal blood vessels. Migratory pattern of the schistosomula of S. japonicum through human skin appeared to be different from
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S. mansoni and S. haematobium. Within 24 h after exposure to infection over 90% of the schistosomula of S. japonicum were found within the dermis and only less than 10% were present in the epidermis. Surprisingly, within 2 h after exposure to infection several of these schistosomula had already entered the dermal blood vessels. These findings thus suggest that schistosomula of S. japonicum unlike schistosomula of S. mansoni and S. haematobium have remarkable ability to penetrate through various layers of human skin with ease, especially the epidermal basement membrane. The presence of several schistosomula within the dermal blood vessels suggest that in human all three schistosomes may be potentially using the blood route to reach the lungs from skin. These findings were similar to those reported previously for S. mansoni in mice (Wheater and Wilson, 1979), and S. japonicum in mice (He et al., 1990). Although dermal blood vessels appears to be the major route of migration into lungs, the parasites can occasionally reach lungs through lymphatic circulation as well (Bayssade-Dufour et al., 1994; Georgi et al., 1987; Lozzi et al., 1996; Mountford et al., 1988). One of the interesting features of S. japonicum migration through human skin is the speed. Studies using a mouse model also show that S. japonicum can migrate faster through the skin (He et al., 1990). Migrating schistosomula of S. japonicum do not to stay in the skin of mice for more than a day. By day 2 after infection schistosomula were present in the lungs (He, 1993) and their arrival in the lungs peaked by day 3 (Gui et al., 1995; Rheinberg et al., 1998). A similar comparison of the migratory pattern of schistosomula of S. mansoni and S. haematobium in mice showed that their numbers peaked in the lungs only by day 6 after infection (Rheinberg et al., 1998). The potential mechanism of the swift migration of S. japonicum through the host skin including human skin is not known. It is possible that the schistosomula of S. japonicum may posses more potent penetrating enzymes or molecules than the other two schistosomes. This might explain the ability of S. japonicum to establish in more than 46 species of nonhuman mammalian hosts (He et al., 2001). The faster migration of S. japonicum through the host tissue is also potentially allowing them reach the predilection site sooner than S. mansoni and S. haematobium and to mature early. S. japonicum oviposition occurs in 24–27 days versus 30–35 for S. mansoni and 60–63 days for S. haematobium (Burden and Ubelaker, 1981; He and Yang, 1980). Differences in the migratory pattern between S. japonicum and the other two species also parallel differences in host responses towards these parasites. Previous studies in mice showed that after entry, schistosomula of S. mansoni remain in the epidermal/dermal layers of the skin for more than 72 h before migrating further into the lungs (Ramaswamy et al., 1997; Wheater and Wilson,
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1979). Host responses in the skin of mice during this time show a subdued inflammatory response in the skin evidenced by fewer infiltrating cells (Ramaswamy et al., 1997), suppression of ICAM-1 and VCAM-1 (Ramaswamy et al., 1998; Trottein et al., 1999), induction of PGE2 and IL-10 in keratinocytes (Ramaswamy et al., 2000), increased production of IL-1ra (Ramaswamy et al., 1995), and suppression of IL-1a, IL-1b, IL-2, and IFN-c (Kumar and Ramaswamy, 1999; Ramaswamy et al., 1995). Therefore, to test whether similar changes are happening in the human skin, we first measured the levels of cytokines (IL-1a, IL-1b, IL-1ra, IL-2, IL-10, IL-12, and IFN-c) in the epidermis after infection of human skin in organ culture. Results from these studies paralleled those observed in the mice suggesting that the schistosomula may be causing similar immunosuppression in the human skin as well. A comparison of the cytokine responses to the three different schistosomes in the human skin showed that all three schistosomes induced production of IL-1ra and IL-10 in the skin. Interestingly, the amount of IL-1ra stimulated by schistosomula of S. japonicum was significantly higher than those induced by the other two species. The resultant anti-inflammatory milieu created around the parasite is believed to form a smoke screen potentially allowing the parasite to escape host detection. Parasite-induced production of these anti-inflammatory cytokines in the human skin appears to increase with time. Above results also suggested that one can measure cytokine changes in the human skin in organ culture by ELISA and RT-PCR after infection with schistosomes. Subsequently, we analyzed a whole spectrum of cytokines in the human skin in organ culture using a macro gene array. Results from this analysis also confirm that IL-10 is increased in the human skin after infection with all three schistosomes. An increase in IL-7 has been reported to occur in the human and mouse skin after infection with S. mansoni (Wolowczuk et al., 1997). The source of these IL-7 was shown to be keratinocytes (Roye et al., 1998). However, we were unable to see any increase in IL-7 in our skin organ cultures at 8 h after infection with any of the three schistosomes, although S. japonicum infection increased transcripts for another IL2 family of cytokine, IL-15. In fact out of 24 different cytokines analyzed in this study only IL-1ra, IL-10, and TNF-a showed significant increases in the human skin after infection with S. mansoni and S. haematobium. Interestingly, after infections with S. japonicum several cytokines were increased in the human skin. We believe that this difference in the cytokine response after S. japonicum infection may be due to its unique migratory pattern through the human skin. Because there are no additional circulating components in the human skin in organ culture, most likely the cytokine response observed reflects the response of resident cells towards the
invading organisms. Further studies will identify the potential cellular sources of these cytokines in the human skin. At least from our previous studies we know that keratinocytes are one of the major sources of IL-1ra and IL-10 in the human skin after infection with S. mansoni (Ramaswamy et al., 2000; Ramaswamy et al., 1995). S. japonicum infection elicited several different cytokines in the human skin including IL-1b, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a. Although significance of the early increases in these cytokines in the skin is not known at this time, increases in IL-6 and TNF-a has been reported to occur in schistosomiasis mansoni in mouse (Angeli et al., 2001a; Khalil et al., 1996; La Flamme et al., 2000) and in human (Marguerite et al., 1999). Increases in IL-2, IL-6, IL-10, IL-18, and TNF-a transcripts suggest that schistosomula of S. japonicum are potentially eliciting a general Th0 type of responses soon after entry into the human skin. In addition to allowing one to study the migratory pattern of schistosomula and cytokine responses in the skin, the present study also shows that the skin organ culture system could be potentially used to evaluate distribution of specific cells in the epidermal layers of the skin by immunocytochemistry. Following infection with S. mansoni there was significant increases in HLA-DRþ cells. One of the major HLA-DRþ cells in the epidermis are the dendritic cells, predominantly Langerhans cells (Messadi et al., 1988). Although we did not identify the morphology of HLA-DRþ cells in our preparations, the typical dendritic appearance (Fig. 2) suggests that these may be Langerhans cells. Interestingly these cells appear to accumulate in areas where schistosomula have entered. Sato et al. (Sato et al., 1997) made similar observations in the guinea pig skin. This focal accumulation of HLA-DRþ cells may suggest initiation of a potential immune response against invading schistosomula of S. mansoni in the human skin. These observations open up new ways to study early immune responses to schistosomes in the human skin. In conclusion, the results presented in this study show that human skin organ culture is a useful surrogate to study early host responses to schistosomes in the skin. We show that using this model one could study the migratory pattern of the parasite through the skin, study changes in the cell distribution and analyze pattern of cytokine responses in the skin.
Acknowledgments The authors would like to thank Dr. Fred Lewis, Biomedical Research Institute, Rockville, Maryland, for the supply of schistosome life cycle stages through NIH-NIAID contract N01-A1-55270. This work was supported by NIH Grant AI 39066 to KR.
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Katz, S.I., 1993. Dohi Memorial Lecture. The skin as an immunological organ: allergic contact dermatitis as a paradigm. Journal of Dermatology 20, 593–603. Khalil, R.M., Hultner, L., Mailhammer, R., Luz, A., Moeller, J., Mohamed, A.A., Omran, S., Dormer, P., 1996. Kinetics of interleukin-6 production after experimental infection of mice with Schistosoma mansoni. Immunology 89, 256–261. Khammo, N., Bartlett, A., Clothier, R.H., Whitfield, P.J., 2002. The attachment of Schistosoma mansoni cercariae to human skin cells. Parasitology 124, 25–30. Kumar, P., Ramaswamy, K., 1999. Vaccination with irradiated cercariae of Schistosoma mansoni preferentially induced the accumulation of interferon-c producing T cells in the skin and skin draining lymph nodes of mice. Parasitology International 48, 109–119. Kusel, J.R., 1970. The penetration of human epidermal sheets by the cercariae of Schistosoma mansoni and the collection of schistosomula. Parasitology 60, 89–96. La Flamme, A.C., MacDonald, A.S., Pearce, E.J., 2000. Role of IL-6 in directing the initial immune response to schistosome eggs. Journal of Immunology 164, 2419–2426. Lozzi, S.P., Machado, C.R., Gerken, S.E., Mota-Santos, T.A., 1996. Involvement of regional lymph nodes after penetration of Schistosoma mansoni cercariae in naive and infected mice. Memorias do Instituto Oswaldo Cruz 91, 491–498. Marguerite, M., Gallissot, M.C., Diagne, M., Moreau, C., Diakkhate, M.M., Roberts, M., Remoue, F., Thiam, A., Decam, C., Rogerie, F., Cottrez, F., Neyrinck, J.L., Butterworth, A.E., Sturrock, R.F., Piau, J.P., Daff, B., Niang, M., Wolowczuk, I., Riveau, G., Auriault, C., Capron, A., 1999. Cellular immune responses of a Senegalese community recently exposed to Schistosoma mansoni: correlations of infection level with age and inflammatory cytokine production by soluble egg antigen-specific cells. Tropical Medicine and International Health 4, 530–543. McKerrow, J.H., 1997. Cytokine induction and exploitation in schistosome infections. Parasitology 115, S107–112. McKerrow, J.H., Jones, P., Sage, H., Pino-Heiss, S., 1985. Proteinases from invasive larvae of the trematode parasite Schistosoma mansoni degrade connective-tissue and basement-membrane macromolecules. Biochemical Journal 231, 47–51. McLaren, D.J., Clegg, J.A., Smithers, S.R., 1975. Acquisition of host antigens by young Schistosoma mansoni in mice: correlation with failure to bind antibody in vitro. Parasitology 70, 67–75. Mei, H., Thakur, A., Schwartz, J., Lo Verde, P.T., 1996. Expression and characterization of glutathione peroxidase activity in the human blood fluke Schistosoma mansoni. Infection and Immunity 64, 4299–4306. Messadi, D.V., Pober, J.S., Murphy, G.F., 1988. Effects of recombinant c-interferon on HLA-DR and DQ expression by skin cells in short-term organ culture. Laboratory Investigation 58, 61–67. Mountford, A.P., Coulson, P.S., Wilson, R.A., 1988. Antigen localization and the induction of resistance in mice vaccinated with irradiated cercariae of Schistosoma mansoni. Parasitology 97, 11–25. Pearce, E.J., Sher, A., 1987. Mechanisms of immune evasion in schistosomiasis. Contributions to Microbiology and Immunology 8, 219–232. Ramaswamy, K., 1998. Dendritic cell migration from the human skin is inhibited by schistosomula of Schistosoma mansoni. In: N.I. Talwar, G.P., Ganguly, N.K., Rao, K.V.S. (Eds.), Proceedings of the 10th International Congress of Immunology. Monduzzi Editore, Bologna, Italy, pp. 1610–1614. Ramaswamy, K., He, Y.X., Salafsky, B., 1997. ICAM-1 and iNOS expression increased in the skin of mice after vaccination with cirradiated cercariae of Schistosoma mansoni. Experimental Parasitology 86, 118–132. Ramaswamy, K., Kumar, P., He, Y.X., 2000. A role for parasiteinduced PGE2 in IL-10-mediated host immunoregulation by skin
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Schistosoma mansoni, S. haematobium, and S. japonicum: early events associated with penetration and migration of schistosomula through human skin Yi-Xun He, Lin Chen, and K. Ramaswamy* Department of Biomedical Sciences, College of Medicine, University of Illinois, Rockford, IL 61107, USA Received 12 February 2002; received in revised form 9 October 2002; accepted 18 February 2003
Abstract Migratory pattern of schistosomula of Schistosoma mansoni, S. haematobium, and S. japonicum through human skin were analyzed in skin organ cultures. These studies showed that the schistosomula of S. mansoni and S. haematobium has similar migratory patterns through human skin. During the first 24 h after infection nearly 90% of S. mansoni and S. haematobium schistosomula were present only in the epidermis. Majority of the schistosomula were found in the dermis only after 48 h and they appear to reach the dermal vessels around 72 h after infection. Migratory pattern of S. japonicum on the other hand was significantly different from the other two species in that over 90% of the parasites had already reached the dermis within the first 24 h and schistosomula were present in the dermal vessels within 2 h after infection. Analysis of the cytokine pattern at 8 h after infection by a macro gene array and RT-PCR analysis showed that out of 24 different cytokines analyzed only IL-1ra, IL-10, and TNF-a were increased in the human skin following infections with S. mansoni and S. haematobium, whereas, after infection with S. japonicum there was significant increases in IL-1b, IL-1ra, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a. Immunohistochemical analysis of epidermal sheets showed focal accumulation of HLA-DRþ cells in areas where schistosomula of S. mansoni had entered the human skin. Ó 2003 Elsevier Science (USA). All rights reserved. Index Descriptors and Abbreviations: Trematode, S. mansoni, S. haematobium, S. japonicum, human skin, schistosomula, migration, cytokines, HLADRþ cells. Interleukin (IL), tumor necrosis factor (TNF), intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
1. Introduction Human schistosomiasis is a water-borne infection caused mainly by three different species of schistosomes, Schistosoma mansoni, S. japonicum, and S. haematobium. Current estimates show that more than 200 million people are infected with these parasites and another 300 million are at risk (WHO, 1993). Human infections occur when cercariae, the infective stages of these parasites, penetrate intact skin and enter into the body (Wheater and Wilson, 1979). This is the only known route of entry for this parasite into human. Yet unfor-
*
Corresponding author. Fax: 1-815-395-5666. E-mail address: [email protected] (K. Ramaswamy).
tunately, very little has been studied on the mechanism of penetration or host response to these parasites in the human skin (Bartlett et al., 2000; Fusco et al., 1993; Khammo et al., 2002; Kusel, 1970) and no information is available on the kinetics of schistosome migration through human skin. To begin to understand the early host responses and migratory pattern of various schistosomula through human skin, we have used a human skin organ culture system (Ramaswamy, 1998) that allowed us to infect human skin pieces ex vivo with cercariae of all three schistosomes and monitor kinetics of schistosomula migration. Once thought to be primarily a target for immune attack during various reactions, the skin, particularly the epidermis, has been shown to actively participate in the generation of immunologic responses affecting the
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00024-9
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skin and the entire immune system (Katz, 1993). Advances in immunodermatology during the past one decade has shown that epidermal cells, specifically keratinocytes, Langerhans cells, and epidermal T lymphocytes can elaborate an array of cytokines into the microenvironment that form a meaningful network of immune responses in the epidermis which is important in the initiation of an appropriate immune responses against invading foreign agents (Uchi et al., 2000). Few studies that have looked to date on the cytokine responses in the mouse skin suggested that the migrating schistosomula are capable of modulating host immune responses in the skin (Angeli et al., 2001b; Ramaswamy et al., 2000; Ramaswamy et al., 1995). At present we do not know whether similar changes are induced in the human skin by all three species of schistosomes. Similarly, we know only very little on the role of epidermal and dermal cells on the host-parasite relationship in human schistosomiasis. In addition to analyzing the migratory pattern of the three schistosomes through skin, the organ culture system has allowed us to compare some of the early cytokine responses associated with parasite entry and migration through the human skin.
2.2. Parasite Biomphalaria glabrata snails infected with S. mansoni and Bulinus truncatus snails infected with S. haematobium were obtained from Dr. Fred Lewis, Biomedical Research Institute, Rockville, MD. S. japonicum life cycle is maintained at our facility in Oncomelania hupensis nosophora snails and ICR strain of mice. Cercariae were collected from the infected snails as described previously (He, 1993; Ramaswamy et al., 1996). In all our studies infections were achieved within 1 h of shedding the cercariae with all three species. 2.3. Histology
2. Materials and methods
Standard paraffin embedded histological procedures were followed for analysis of human skin pieces. Migratory behavior of schistosomula in the human skin in organ cultures was evaluated by staining 6–7 lm serial sections of the skin pieces with hematoxylin and eosin. Number of parasites present in 150–230 consecutive serial sections (which accounted for 1 to 1.5 mm thickness) from each skin piece were counted and the total number of schistosomula present in the epidermis and dermis were determined. Care was taken not to count the same parasite in adjacent sections. Values are presented as percent recovery in epidermis and dermis.
2.1. Human skin organ culture
2.4. Immunohistochemistry
Fresh human foreskin samples collected from SwedishAmerican Hospital, Rockford, USA, was used in these experiments. The use of human skin in these studies were approved by the Institutional Review Board of the College of Medicine at Rockford, University of Illinois. Organ cultures were prepared as described previously (Ramaswamy, 1998). Briefly, fresh foreskin samples cut approximately into 1.5 cm3 cubes were kept in air/liquid biphasic cultures containing RPMI 1640, 5% FBS, 5% human AB serum or 10% fetal bovine serum in some studies, 10 lg/ml gentamicin, and 25 lg/ml fungizone (Gibco, Grand Island, NY) at 37 °C and 5% CO2 in air. Infection was achieved by placing 150–300 cercariae of S. mansoni, S. haematobium, or S. japonicum suspended in 50–100 ll of sterile water under a cover glass directly on to the epidermal surface of the skin and incubated for 60 min at 37 °C. Skin pieces were usually infected within 1–4 h after collection. Following exposures to the parasites the skin pieces were briefly rinsed in RPMI media and incubated further for varying periods of time (1–72 h) in fresh media. Skin pieces from the same individual cultured simultaneously, but not infected with schistosome cercariae, were used as negative controls. Samples for histology were all fixed in buffered formalin. Skin samples were also snap frozen in liquid nitrogen for RT-PCR analysis.
Distribution of Class II major histocompatibility antigen expressing cells were examined in the epidermal sheets of 15 different individuals 48 h after exposure to infection with S. mansoni. Epidermal sheets were prepared by incubating skin pieces in 0.5 M ammonium thiocyanate (Sigma, St. Louis, MO) for 30 min at 37 °C. This allowed the epidermal layer to be peeled off easily. Uninfected skin pieces from the same individual were used as negative controls. After removing the epidermal sheet they were divided into two and mounted on to 2% aminopropyltriethoxysilane (Sigma) coated slides with dermal side up. One of these pieces remained as negative staining control. Epidermal sheets were then washed three times with phosphate-buffered saline (pH 7.3), fixed in acetone for 10 min at room temperature, air dried, and stored at )70 °C until used. Before immunohistochemical staining, slides were thawed to room temperature and epidermal sheets were incubated in a peroxidase blocking solution (Pierce chemicals, Rockford, IL) for 10 min at room temperature. Five percent normal rat serum was used to block non-specific binding sites. To identify Class II positive cells, epidermal sheets were incubated with a mouse anti-human HLA-DR monoclonal antibody (IgG2a , Pharmingen, San Diego, CA) for 1 h at room temperature. Horseradish peroxidase labeled rat anti mouse
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IgG2a antibodies (Pharmingen) were used as the secondary antibody. Color was developed using a metal enhanced diaminobenzidine substrate (Pierce Chemicals) and the epidermal sheets were counter stained with MayerÕs hematoxylin (Fisher Chemicals, Pittsburgh, PA) to identify HLA-DR positive cells. Control samples treated similarly were incubated with a non-specific mouse IgG2a antibody (Pharmingen). 2.5. Cytokine analysis Secreted levels of cytokines for IL-1a, IL-1b, IL1ra, IL-2, IL-10, IL-12, and IFN-c were evaluated in the human epidermal sheets 48 h after exposure to infections with S. mansoni. Briefly, epidermal sheets were peeled off as detailed above, homogenized in a sonicator (Fisher Scientific) in phosphate-buffered saline (pH 7.0) over ice, and centrifuged at 10,000 rpm to collect the soluble proteins in the homogenate. Total protein concentration in the supernatant was estimated using a protein assay kit (BCA kit, Pierce Chemicals). All protein samples were normalized to the same concentration by diluting with PBS. Level of protein in the skin samples varied from 1.5 mg to 2.7 mg/ml. Levels of cytokine in the epidermal cell homogenate was determined using an ELISA using kits purchased from R&D Systems (Minneapolis, MN). Uninfected skin samples from the same individual cultured similarly were used as controls. Ten to 15 foreskin samples were used per group in each experiment. 2.6. RT-PCR analysis of cytokine for IL-1b, IL-1ra, and IL-10 in human skin Human foreskin samples were cultured with 150 cercariae of S. mansoni, S. haematobium, or S. japonicum in RPMI-1640 medium with 10% FBS or in media alone for 4 and 8 h at 37 °C with 5% CO2 . At each time point after culture, skin samples were collected and total RNA extracted with TRIzol reagent (Life Technologies, Rockvill, MD) for reverse transcription using RETROscript (Ambion, Austin, TX). The cDNA of b-actin in each sample was first PCR amplified (Perkin–Elmer, Norwalk, CT) using b-actin-specific primers (Ambion). Based on the band density of b-actin, each sample was then adjusted to approximately the same level. Individual samples were then PCR amplified for IL-1b, IL-1ra, and IL-10, using gene-specific primers for IL-1b (Ambion), IL10 (Ambion), and IL-1ra (Chan et al., 1992). Primers for b-actin, IL-1b, IL-1ra, and IL-10 amplify 294-, 238-, 492-, and 297-bp target fragments, respectively. PCRs were performed as follows: for b-actin, 3 min at 94 °C, 30 s at 55 °C, and 30 s at 72 °C for 30 cycles; for IL-1b and IL-10, 3 min at 94 °C, 30 s at 57 °C, and 30 s at 72 °C for 30 cycles; for IL-1ra, 3 min at 94 °C, 30 s at 60 °C, and 30 s at 72 °C for 30 cycles. The final
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elongation was followed by 5 min at 72 °C for all target cDNA amplifications. The products were resolved on a 1.5% agarose gel and stained with ethidium bromide. Photographs of gels were scanned and band densities were analyzed using NIH Image software. Results are expressed as target band intensity divided by b-actin band density. 2.7. Macro gene array analysis of cytokines in human skin after exposure to infection with schistosomes A macro gene array was performed using a NonradGEArray kit (Catalog # hGEA9912090) purchased from Superarray (Bethesda, MD) to detect cytokine mRNA expression in human skin 8 h after exposure to 150 cercariae of S. mansoni, S. haematobium, or S. japonicum for 8 h. The assay was performed according to the instructions provided by the manufacturer. Cytokine genes analyzed by this gene array included IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL11, IL-12 (p35), IL-12 (p40), IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN-c, TNF-a, and TNF-b. b-Actin and GAPDH were used as controls. Briefly, total RNA extracted with TRIzol was used as a template for synthesis of biotinylated probes using reverse transcription. Membranes spotted with specific cytokine cDNA fragments were then hybridized with these labeled probes. Signal was developed after incubating the membranes with alkaline phosphatase conjugated streptavidin and alkaline phosphatase chemiluminescence substrate. Signal density in each spot was then scanned and analyzed using an NIH Image software. Results are expressed as target band intensity divided by b-actin band density. Experiments were repeated twice using five skin samples per group. 2.8. Statistical analysis Statistical significance of the means of values was tested by one-way analysis of variance using Sigmastat program (Jandel Scientific, San Rafael, CA). All pairwise multiple comparison was done using Student– Newman–Keuls method. Probability value of 1% or lower was considered significant. All values are expressed as means SD.
3. Results 3.1. Migratory pattern of schistosomula through human skin Histological analysis of serial section of human skin samples exposed to 150 cercariae of S. mansoni showed migrating parasites in various layers of the skin at different intervals after exposure to infection. Fig. 1 shows
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Fig. 1. Histological analysis of the migration of schistosomula of S. mansoni (A–G), S. haematobium (I), and S. japonicum (J–L) through human skin in organ cultures. Parasite migration was traced by analyzing 6–7 lm serial sections of the skin pieces. At 4 h after exposure to infection (A), transformed schistosomula of S. mansoni were seen in the outermost layers of the epidermis. By 8 h (B) they had already penetrated deeper into the epidermis and lay horizontal to the basement membrane. Sections examined at 12 h (C) and 16 h (D) after infection showed schistosomula of S. mansoni in the process of penetrating through the basement membrane. By 24 h (E) some of the parasites have already entered the dermis. The schistosomula then migrated further deeper into the dermis by 48 h (F) and reached close to the blood vessels by 72 h (G). Schistosomula of S. japonicum were seen in the dermal vessels (J–L) within 2 h after exposure to infection. However, it took more than 8 h for schistosomula of S. haematobium to cross the epidermal basement membrane (I). Migratory pattern of the schistosomula of S. haematobium through human skin closely resembled those of S. mansoni. Section of uninfected skin (H) prepared at 72 h after culture remained as control. All sections were stained with H&E. Original magnification was X100. Arrows (!) indicate cut sections of schistosomulum. Sections shown are from one of 10 similarly infected human skin pieces.
sequence of events in the migration of schistosomula of S. mansoni, S. japonicum, and S. haematobium through the human skin. During the initial 4 h after exposure to S. mansoni infection, transformed schistosomula were seen in the cuticle and outermost layers of the epidermis
(Fig. 1A). At 8 h after exposure to infection (Fig. 1B) 94% of the schistosomula of S. mansoni were present in the epidermis. Interestingly most of the parasites lay horizontal to the basement membrane before entering into the dermis. Skin sections examined at 12 h (Fig. 1C)
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and 16 h (Fig. 1D) after exposure to S. mansoni infection showed parasites in the process of penetrating through the basement membrane into the dermis. By 24 h (Fig. 1E) only 11% of the parasites had entered the dermis, still 89% were in the epidermis. However, once in the dermis schistosomula of S. mansoni appeared to move faster and deeper (Fig. 1F) until they reached closer to the blood vessels. Most of the parasites observed at 72 h after infection were either inside or closer to the blood vessels in the hypodermis (Fig. 1G). Migratory pattern of S. haematobium schistosomula through human skin appeared to be similar to those of S. mansoni schistosomula. Over 93% of schistosomula of S. haematobium were seen only in the epidermis up to 8 h after exposure to infection (Fig. 1-I). Similar to S. mansoni, the schistosomula of S. haematobium also appear to enter the dermis in large numbers around 48 h and reach closer to the dermal vessels around 72 h after exposure to the infection. Compared to S. mansoni and S. haematobium, schistosomula of S. japonicum appeared to migrate faster through human skin. At 8 h after exposure to infection 73% of the parasite had already migrated to the dermis and by 24 h over 90% of the parasites had reached dermis. Migrating schistosomula of S. japonicum were seen inside dermal blood vessels as early as 2 h after exposure to the infection (Fig. 1J–L). 3.2. HLA-DR expression in human skin after exposure to infection with S. mansoni Immunohistochemical staining of normal human skin showed that HLA-DRþ cells are randomly distributed in the epidermis of control skin (Fig. 2A). However, 48 h after exposure to infection with S. mansoni, there was an increased focal accumulation of HLA-DRþ cells in ar-
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eas where schistosomula has entered into the epidermis (Fig. 2B). 3.3. Levels of cytokine protein in human epidermal sheets after exposure to infection with S. mansoni Previous studies using a human keratinocyte cell line showed significant increases in the expression of IL-1ra when ES products from schistosomula of S. mansoni were added to the cultures (Ramaswamy et al., 1995). Studies in mice showed that in addition to IL-1ra there was also an increase in IL-10 in the skin following infection with S. mansoni (Ramaswamy et al., 2000). Analysis of human skin in organ culture showed a similar increase in IL-1ra and IL-10 within 48 h after exposure to infection with S. mansoni (Fig. 3). Levels of IL-1a, IL-1b, IL-2, IL-12, and
Fig. 3. Analysis of different cytokine levels in human epidermal homogenate 48 h after infection with S. mansoni showed significant increases in the levels of IL-1ra and IL-10 but not in IL-1a, IL-1b, IL-2, IL-12, or IFN-c. Epidermal sheets were prepared as described in Section 2 and homogenized. After normalizing the samples to similar protein concentration, the levels of cytokine in the epidermal cell homogenate were determined by an ELISA using respective kits for each cytokine. Data represent one of three similar experiments using skin pieces from 10 individuals. Control skin samples used were from the same individual. *Significantly different from controls (p < 0:01).
Fig. 2. Distribution of HLA-DRþ cells in human epidermal sheets after infection with S. mansoni (B). Samples of human skin were exposed to 150– 300 cercariae of S. mansoni ex vivo in organ cultures. At 48 h after infection, epidermal sheets were peeled off, fixed in acetone and incubated with mouse anti-human HLA-DR monoclonal antibody (IgG2a ) for 1 h at room temperature. HRP-labeled rat anti-mouse IgG2a and DAB substrate were used to identify positive cells. MayerÕs hematoxylin was used as counterstain. Figures shown are representative of one of 15 different epidermal sheets analyzed. Uninfected epidermal sheet samples collected from the same individual remained as controls (A). Original magnification was 100.
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Fig. 5. Cytokine gene-expression pattern in human skin after infection with S. mansoni, S. haematobium, or S. japonicum. A macro gene array analysis was performed for 23 different cytokines in the human skin 8 h after infection with the three schistosomes in organ culture. After developing the signals, density of each spot was analyzed using an NIH image software and results are expressed as target band density divided by b-actin band density for each sample. Only cytokines that showed significant differences from controls are presented in the graph. These experiments were repeated two times using skin samples from five individuals with similar results. Data presented are from one of these experiments. * and ** significantly different, p < 0:05 and p < 0:01 respectively compared with control. Fig. 4. Analysis of cytokine transcripts for IL-1b, IL-1ra, and IL-10 in human foreskin samples exposed to 150 cercariae of S. mansoni, S. haematobium, or S. japonicum. Total RNA was extracted from skin samples collected at 4 or 8 h after infection and reverse transcribed. Individual samples were then PCR amplified using gene-specific primers. The PCR product were resolved on a 1.5% agarose gel and stained with ethidium bromide (A). Images were then scanned and ratio of target density to b-actin was calculated (B). Data represent one of three similar experiment using skin samples from five to seven individuals per group. *Significantly different from controls ðp < 0:01Þ.
IFN-c did not show any significant difference from control skin samples at this time point (Fig. 3). 3.4. Message levels for IL-1ra and IL-10 in human skin after exposure to infection with S. mansoni, S. haematobium, or S. japonicum We then confirmed the above observation by measuring levels of cytokine mRNA in the skin organ cultures after exposure to infection with S. mansoni. These studies showed that message levels for IL-1ra and IL-10 is significantly increased in the skin by 8 h after exposure to infection with S. mansoni (Fig. 4A). A comparison of the cytokine responses in the skin after exposure to infection with the other two species of schistosomes showed that a similar increase in IL-1ra and IL-10 occurred in the human skin after infections with S. japonicum and S. haematobium (Fig. 4A). IL-1b transcript levels did not show any significant difference from control skin samples (Fig. 4B). Substantially higher levels of IL-1ra transcripts were found in the human skin after exposure to infection with S. japonicum than S. mansoni or S. haematobium. Similarly, compared to 4 h the skin samples collected at 8 h after exposure to infection with all three schistosomes had higher levels of IL-1ra and IL-10 transcripts (Fig. 4B).
3.5. Macro gene array analysis of human skin for cytokine expression after exposure to infection with S. mansoni, S. haematobium, or S. japonicum Given that there are significant differences in the levels of IL-1ra and IL-10 in the human skin in organ culture after infection with all three schistosomes, we then wanted to analyze whether there is any significant difference in the expression of other cytokines as well. We used a macro gene array to analyze message levels of 23 different cytokines (Fig. 5). These studies showed that 8 h after exposure to infection with S. mansoni there was a significant decrease in IL-1a, whereas, IL-10 and TNF-a showed a significant increase. A similar decrease in IL-1a was also evident after exposure to infection with S. japonicum. However, following S. japonicum infection there was a significant increase in the message levels for IL-1b, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a in the human skin. Pattern of cytokine message in the human skin after exposure to infection with S. haematobium was apparently similar to those after S. mansoni infection. However, IL-1a levels did not show any significant changes after exposure to infection with S. haematobium. Compared to control skin there was no significant change in IL-3, IL-4, IL-5, IL-7, IL-9, IL-11, IL-12, IL-13, IL-14, IL-16, IL-17, IFN-c, and TNF-b in the infected skin pieces (data not shown).
4. Discussion Schistosomiasis is still a major human health hazard in many tropical and subtropical areas of the world (WHO, 1993). Human infections occur when cercariae, the infective stages of the parasite, penetrate intact skin and
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enter into the body. Thus, skin appears to be the primary organ of initial contact between the parasite and the human host. Traditionally a mouse model is used to study schistosome–host interaction. Using this model substantial progress has been made during the past four decades in unraveling the mechanism of skin penetration and immunobiology of schistosomiasis in the murine skin (Gui et al., 1995; He, 1993; Ramaswamy et al., 1997; Salafsky et al., 1984). Yet there remains a gap in our understanding of the host–parasite relationship of the three schistosomes in the human skin. In the present study using a human skin organ culture system we have compared the migratory pattern of the schistosomula of S. mansoni, S. haematobium, and S. japonicum, evaluated changes in the cytokine pattern following infection and examined alterations in the distribution of specific cells in the skin as a result of infection. Analysis of the migratory pattern of schistosomula showed that all three species of schistosomes were capable of penetrating human skin in organ culture and were able to migrate through various layers of the skin in a predictable manner. Proteolytic enzymes present in the acetabular glands are believed to help in this process of skin penetration and entry (Dresden et al., 1977; McKerrow et al., 1985; Salter et al., 2000; Stirewalt, 1982). It is often thought that schistosomula find their way into the skin mainly through hair follicles (Stirewalt, 1982). However, our studies suggest that schistosomula of all three species can penetrate and enter into human skin through any skin surface irrespective of hair follicle areas as has been reported in gerbils (BayssadeDufour et al., 1994) and rhesus monkeys (He et al., 1992). Although entry of all three species of schistosomula into human epidermis occurs within minutes, there appears to be significant differences in their migratory pattern through the skin. Schistosomula of S. mansoni and S. haematobium showed apparently similar migratory pattern through human skin. After entering into the skin majority of the schistosomula of S. mansoni and S. haematobium remains in the epidermis for about 24 h before traversing the basement membrane. The reason for such a long stay in the epidermis is not clear. However, it has been postulated that during this time schistosomula may be acquiring host molecules on their surface (Dessein et al., 1981; McLaren et al., 1975; Ruppel et al., 1984; Sher and Benno, 1982) or are developing strategy (Hawn and Strand, 1993; Mei et al., 1996) that potentially allow them to evade host responses (McKerrow, 1997; Pearce and Sher, 1987). Once after crossing the basement membrane they appeared to migrate faster aiming toward the dermal blood vessels. After entering the skin it takes nearly three days for the schistosomula of S. mansoni and S. haematobium to reach dermal blood vessels. Migratory pattern of the schistosomula of S. japonicum through human skin appeared to be different from
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S. mansoni and S. haematobium. Within 24 h after exposure to infection over 90% of the schistosomula of S. japonicum were found within the dermis and only less than 10% were present in the epidermis. Surprisingly, within 2 h after exposure to infection several of these schistosomula had already entered the dermal blood vessels. These findings thus suggest that schistosomula of S. japonicum unlike schistosomula of S. mansoni and S. haematobium have remarkable ability to penetrate through various layers of human skin with ease, especially the epidermal basement membrane. The presence of several schistosomula within the dermal blood vessels suggest that in human all three schistosomes may be potentially using the blood route to reach the lungs from skin. These findings were similar to those reported previously for S. mansoni in mice (Wheater and Wilson, 1979), and S. japonicum in mice (He et al., 1990). Although dermal blood vessels appears to be the major route of migration into lungs, the parasites can occasionally reach lungs through lymphatic circulation as well (Bayssade-Dufour et al., 1994; Georgi et al., 1987; Lozzi et al., 1996; Mountford et al., 1988). One of the interesting features of S. japonicum migration through human skin is the speed. Studies using a mouse model also show that S. japonicum can migrate faster through the skin (He et al., 1990). Migrating schistosomula of S. japonicum do not to stay in the skin of mice for more than a day. By day 2 after infection schistosomula were present in the lungs (He, 1993) and their arrival in the lungs peaked by day 3 (Gui et al., 1995; Rheinberg et al., 1998). A similar comparison of the migratory pattern of schistosomula of S. mansoni and S. haematobium in mice showed that their numbers peaked in the lungs only by day 6 after infection (Rheinberg et al., 1998). The potential mechanism of the swift migration of S. japonicum through the host skin including human skin is not known. It is possible that the schistosomula of S. japonicum may posses more potent penetrating enzymes or molecules than the other two schistosomes. This might explain the ability of S. japonicum to establish in more than 46 species of nonhuman mammalian hosts (He et al., 2001). The faster migration of S. japonicum through the host tissue is also potentially allowing them reach the predilection site sooner than S. mansoni and S. haematobium and to mature early. S. japonicum oviposition occurs in 24–27 days versus 30–35 for S. mansoni and 60–63 days for S. haematobium (Burden and Ubelaker, 1981; He and Yang, 1980). Differences in the migratory pattern between S. japonicum and the other two species also parallel differences in host responses towards these parasites. Previous studies in mice showed that after entry, schistosomula of S. mansoni remain in the epidermal/dermal layers of the skin for more than 72 h before migrating further into the lungs (Ramaswamy et al., 1997; Wheater and Wilson,
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1979). Host responses in the skin of mice during this time show a subdued inflammatory response in the skin evidenced by fewer infiltrating cells (Ramaswamy et al., 1997), suppression of ICAM-1 and VCAM-1 (Ramaswamy et al., 1998; Trottein et al., 1999), induction of PGE2 and IL-10 in keratinocytes (Ramaswamy et al., 2000), increased production of IL-1ra (Ramaswamy et al., 1995), and suppression of IL-1a, IL-1b, IL-2, and IFN-c (Kumar and Ramaswamy, 1999; Ramaswamy et al., 1995). Therefore, to test whether similar changes are happening in the human skin, we first measured the levels of cytokines (IL-1a, IL-1b, IL-1ra, IL-2, IL-10, IL-12, and IFN-c) in the epidermis after infection of human skin in organ culture. Results from these studies paralleled those observed in the mice suggesting that the schistosomula may be causing similar immunosuppression in the human skin as well. A comparison of the cytokine responses to the three different schistosomes in the human skin showed that all three schistosomes induced production of IL-1ra and IL-10 in the skin. Interestingly, the amount of IL-1ra stimulated by schistosomula of S. japonicum was significantly higher than those induced by the other two species. The resultant anti-inflammatory milieu created around the parasite is believed to form a smoke screen potentially allowing the parasite to escape host detection. Parasite-induced production of these anti-inflammatory cytokines in the human skin appears to increase with time. Above results also suggested that one can measure cytokine changes in the human skin in organ culture by ELISA and RT-PCR after infection with schistosomes. Subsequently, we analyzed a whole spectrum of cytokines in the human skin in organ culture using a macro gene array. Results from this analysis also confirm that IL-10 is increased in the human skin after infection with all three schistosomes. An increase in IL-7 has been reported to occur in the human and mouse skin after infection with S. mansoni (Wolowczuk et al., 1997). The source of these IL-7 was shown to be keratinocytes (Roye et al., 1998). However, we were unable to see any increase in IL-7 in our skin organ cultures at 8 h after infection with any of the three schistosomes, although S. japonicum infection increased transcripts for another IL2 family of cytokine, IL-15. In fact out of 24 different cytokines analyzed in this study only IL-1ra, IL-10, and TNF-a showed significant increases in the human skin after infection with S. mansoni and S. haematobium. Interestingly, after infections with S. japonicum several cytokines were increased in the human skin. We believe that this difference in the cytokine response after S. japonicum infection may be due to its unique migratory pattern through the human skin. Because there are no additional circulating components in the human skin in organ culture, most likely the cytokine response observed reflects the response of resident cells towards the
invading organisms. Further studies will identify the potential cellular sources of these cytokines in the human skin. At least from our previous studies we know that keratinocytes are one of the major sources of IL-1ra and IL-10 in the human skin after infection with S. mansoni (Ramaswamy et al., 2000; Ramaswamy et al., 1995). S. japonicum infection elicited several different cytokines in the human skin including IL-1b, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-a. Although significance of the early increases in these cytokines in the skin is not known at this time, increases in IL-6 and TNF-a has been reported to occur in schistosomiasis mansoni in mouse (Angeli et al., 2001a; Khalil et al., 1996; La Flamme et al., 2000) and in human (Marguerite et al., 1999). Increases in IL-2, IL-6, IL-10, IL-18, and TNF-a transcripts suggest that schistosomula of S. japonicum are potentially eliciting a general Th0 type of responses soon after entry into the human skin. In addition to allowing one to study the migratory pattern of schistosomula and cytokine responses in the skin, the present study also shows that the skin organ culture system could be potentially used to evaluate distribution of specific cells in the epidermal layers of the skin by immunocytochemistry. Following infection with S. mansoni there was significant increases in HLA-DRþ cells. One of the major HLA-DRþ cells in the epidermis are the dendritic cells, predominantly Langerhans cells (Messadi et al., 1988). Although we did not identify the morphology of HLA-DRþ cells in our preparations, the typical dendritic appearance (Fig. 2) suggests that these may be Langerhans cells. Interestingly these cells appear to accumulate in areas where schistosomula have entered. Sato et al. (Sato et al., 1997) made similar observations in the guinea pig skin. This focal accumulation of HLA-DRþ cells may suggest initiation of a potential immune response against invading schistosomula of S. mansoni in the human skin. These observations open up new ways to study early immune responses to schistosomes in the human skin. In conclusion, the results presented in this study show that human skin organ culture is a useful surrogate to study early host responses to schistosomes in the skin. We show that using this model one could study the migratory pattern of the parasite through the skin, study changes in the cell distribution and analyze pattern of cytokine responses in the skin.
Acknowledgments The authors would like to thank Dr. Fred Lewis, Biomedical Research Institute, Rockville, Maryland, for the supply of schistosome life cycle stages through NIH-NIAID contract N01-A1-55270. This work was supported by NIH Grant AI 39066 to KR.
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