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Aloe Barbadensis Extracts Reduce the Production of Interleukin-10 After Exposure to Ultraviolet Radiation

1998; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1998.00181.x

1523-1747

Son Won Byeon, Ronald P. Pelley, Stephen E. Ullrich, Todd A. Waller, Corazon D. Bucana, Faith M. Strickland,

Chromatography in Natural Products

Cutaneous exposure to ultraviolet radiation suppresses the induction of T cell mediated responses such as contact and delayed type hypersensitivity (DTH) by altering the function of immune cells in the skin and causing the release of immunoregulatory cytokines. Extracts of crude Aloe barbadensis gel prevent this photosuppression. Because the regulation of contact hypersensitivity and DTH responses differ, we investigated whether protection was afforded by a single or multiple agents in Aloe and the mechanism by which this material prevents suppression of DTH immunity. The ability of Aloe gel to prevent suppression of contact hypersensitivity responses to hapten decayed rapidly after manufacture. In contrast, agents that protected against systemic suppression of DTH responses to Candida albicans were stable over time. Oligosaccharides prepared from purified Aloe polysaccharide prevented suppression of DTH responses in vivo and reduced the amount of IL-10 observed in ultraviolet irradiated murine epidermis. To assess the effect of Aloe extracts on keratinocytes, Pam 212 cells were exposed in vitro to ultraviolet radiation and treated for 1 h with Aloe oligosaccharides. Culture supernatants were collected 24 h later and injected into mice. Supernatants from ultraviolet irradiated keratinocytes suppressed the induction of DTH responses, whereas Aloe oligosaccharide treatment reduced IL-10 and blocked the suppressive activity of the supernatants. These results indicate that Aloe contains multiple immunoprotective factors and that Aloe oligosaccharides may prevent ultraviolet induced suppression of DTH by reducing keratinocyte derived immunosuppressive cytokines. Cutaneous exposure to ultraviolet radiation suppresses the induction of T cell mediated responses such as contact and delayed type hypersensitivity (DTH) by altering the function of immune cells in the skin and causing the release of immunoregulatory cytokines. Extracts of crude Aloe barbadensis gel prevent this photosuppression. Because the regulation of contact hypersensitivity and DTH responses differ, we investigated whether protection was afforded by a single or multiple agents in Aloe and the mechanism by which this material prevents suppression of DTH immunity. The ability of Aloe gel to prevent suppression of contact hypersensitivity responses to hapten decayed rapidly after manufacture. In contrast, agents that protected against systemic suppression of DTH responses to Candida albicans were stable over time. Oligosaccharides prepared from purified Aloe polysaccharide prevented suppression of DTH responses in vivo and reduced the amount of IL-10 observed in ultraviolet irradiated murine epidermis. To assess the effect of Aloe extracts on keratinocytes, Pam 212 cells were exposed in vitro to ultraviolet radiation and treated for 1 h with Aloe oligosaccharides. Culture supernatants were collected 24 h later and injected into mice. Supernatants from ultraviolet irradiated keratinocytes suppressed the induction of DTH responses, whereas Aloe oligosaccharide treatment reduced IL-10 and blocked the suppressive activity of the supernatants. These results indicate that Aloe contains multiple immunoprotective factors and that Aloe oligosaccharides may prevent ultraviolet induced suppression of DTH by reducing keratinocyte derived immunosuppressive cytokines. contact hypersensitivity Excessive exposure to ultraviolet (UV) radiation is harmful, causing sunburn, premature aging of the skin, and mutations leading to skin cancer (Urbach, 1978Urbach F. Evidence and epidermiology of ultraviolet-induced cancers in man.Natandrsquo;l Cancer Inst Monogr. 1978; 50: 5PubMed Google Scholar; Harber and Bickers, 1989Harber L.C. Bickers D.R. Photosensitivity Diseases. 2nd edn. BC Decker, Toronto1989: 112Google Scholar). Studies in laboratory animals have shown that UV radiation in the "B" range (280–320 nm) of the electromagnetic spectrum also contributes to the growth of highly antigenic skin cancers by suppressing T cell mediated immune responses (Kripke, 1974Kripke M.L. Antigenicity of murine skin tumors induced by UV light.J Natl Cancer Inst. 1974; 53: 1333PubMed Google Scholar; Fisher and Kripke, 1977Fisher M.S. Kripke M.L. Systemic alteration induced in mice by ultraviolet light irradiation and its relationship to ultraviolet carcinogenesis.Proc Natl Acad Sci USA. 1977; 74: 1688Crossref PubMed Scopus (353) Google Scholar). Model systems established to study the link between UV induced immune suppression and tumor development showed that cutaneous exposure to low (suberythemal) doses of UV radiation inhibits the induction of contact hypersensitivity (CHS) response to hapten applied at the site of irradiation (Toews et al., 1980Toews G.B. Bergstresser P.R. Streilein J.W. Epidermal Langerhans cell density deermines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.J Immunol. 1980; 124: 445PubMed Google Scholar). Higher doses of UVB radiation induce systemic suppression of CHS responses to hapten applied to unirradiated sites and delayed type hypersensitivity (DTH) responses to infectious agents, including herpes simplex virus, leishmania, Candida albicans, and mycobacteria (Giannini, 1986Giannini M.S.H. Suppression of pathogenesis in cutaneous leishmaniasis by UV-irradiation.Infect Immunol. 1986; 51: 838PubMed Google Scholar; Otani and Mori, 1987Otani T. Mori R. The effects of ultraviolet irradiation of the skin on herpes simplex virus infection: Alteration in immune function mediated by epidermal cells and in the course of infection.Arch Virol. 1987; 96: 1Crossref PubMed Scopus (34) Google Scholar; Denkins et al., 1989Denkins Y.D. Fidler I.J. Kripke M.L. Exposure of mice to UV-B radiation suppresses delayed hypersensitivity to Candida albicans..Photochem Photobiol. 1989; 49: 615Crossref PubMed Scopus (72) Google Scholar; Jeevan and Kripke, 1989Jeevan A. Kripke M.L. Effect of a single exposure to UVB radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice.J Immunol. 1989; 143: 2837PubMed Google Scholar). Susceptibility to UV induced immune suppression of CHS responses may be a risk factor for the development of skin cancer in humans (Yoshikawa et al., 1990Yoshikawa T. Rae V. Bruins-Slot W. Van den Berg J.-W. Taylor J.R. Streilein J.W. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in humans.J Invest Dermatol. 1990; 95: 530Abstract Full Text PDF PubMed Google Scholar). Whereas the precise manner by which UV suppresses T cell immunity is still unclear, CHS and DTH responses appear to be suppressed by different mechanisms (Kripke and Morison, 1986Kripke M.L. Morison W.L. Studies on the mechanism of systemic suppression of contact hypersensitivity by UVB radiation. II. Differences in the suppression of delayed and contact hypersensitivity.J Invest Dermatol. 1986; 86: 543Crossref PubMed Scopus (34) Google Scholar; Kim et al., 1990Kim T.-Y. Kripke M.L. Ullrich S.E. Immunosuppression by factors released from UV irradiated epidermal cells: selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation.J Invest Dermatol. 1990; 94: 26Abstract Full Text PDF PubMed Google Scholar; Rivas and Ullrich, 1994Rivas J.M. Ullrich S.E. The role of IL-4, IL-10, and TNF-α in the immune suppression induced by ultraviolet radiation.J Leuk Biol. 1994; 56: 769PubMed Google Scholar). Suppression of CHS responses involves alterations in Langerhans cell functions and the release of soluble factors such as tumor necrosis factor-α, interleukin (IL)-1, prostaglandin-E2, and cis-urocanic acid (Chung et al., 1986Chung H.T. Burnhan D.K. Robertson B. Roberts L.K. Daynes R.A. Involvement of prostaglandins in the immune alterations caused by the exposure of mice to ultraviolet radiation.J Immunol. 1986; 137: 2478PubMed Google Scholar; Robertson et al., 1987Robertson B. Gahring L. Newton R. Daynes R.A. In vivo administration of IL-1 to normal mice depresses their capacity to elicit contact hypersensitivity responses: prostaglandins are involved in this modification of immune function.J Invest Dermatol. 1987; 88: 380Abstract Full Text PDF PubMed Google Scholar; Harriott-Smith and Haliday, 1988Harriott-Smith T.G. Haliday W.J. Suppression of contact hypersensitivity by short term ultraviolet irradiation. II. The role of urocanic acid.Clin Exp Med. 1988; 72: 174Google Scholar; Vermeer and Streilein, 1990Vermeer M. Streilein J.W. Ultraviolet B light-induced alterations in epidermal Langerhans cells are mediated in part by tumor necrosis factor-alpha.Photodermatol Photoimmunol Photomed. 1990; 7: 258PubMed Google Scholar; Simon et al., 1991Simon J.C. Tigelaar R.E. Bergstresser P.R. Edelbaum D. Cruz P.D. Ultraviolet B radiation converts Langerhans cells from immunogenic to tolerogenic antigen-presenting cells.J Immunol. 1991; 146: 485PubMed Google Scholar). Recent studies have demonstrated that UV induced keratinocyte derived IL-10 systemically suppresses DTH responses to alloantigen by downregulating antigen presenting cell functions and triggering the formation of antigen specific suppressor T cells (Noonan and De Fabo, 1992Noonan F.P. De Fabo E.C. Immunosuppression by ultraviolet B radiation: initiation by urocanic acid.Immunol Today. 1992; 13: 250Abstract Full Text PDF PubMed Scopus (243) Google Scholar; Ullrich, 1994Ullrich S.E. Mechanisms involved in the systemic suppression of antigen-presenting cell function by UV irradiation: keratinocyte-derived IL-10 modulate antigen-presenting cell function of splenic adherent cells.J Immunol. 1994; 152: 3410PubMed Google Scholar). A central role for IL-10 in mediating suppression of DTH responses has been shown using neutralizing anti-IL10 antibody (Rivas and Ullrich, 1994Rivas J.M. Ullrich S.E. The role of IL-4, IL-10, and TNF-α in the immune suppression induced by ultraviolet radiation.J Leuk Biol. 1994; 56: 769PubMed Google Scholar). Injection of the antibody into UV irradiated mice blocks the suppression of DTH responses. Furthermore, absorbtion of supernatants from UV irradiated keratinocyte cultures with anti-IL-10 antibody removes their in vivo suppressive activity. The role of IL-10 in the suppression of CHS immunity is less clear because injection of the cytokine blocks the induction of CHS responses in mice, but the treatment of UV irradiated mice with anti-IL-10 antibody fails to restore their CHS responses (Rivas and Ullrich, 1994Rivas J.M. Ullrich S.E. The role of IL-4, IL-10, and TNF-α in the immune suppression induced by ultraviolet radiation.J Leuk Biol. 1994; 56: 769PubMed Google Scholar; Niizeki and Streilein, 1997Niizeki H. Streilein J.W. Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin is mediated via interleukin-10.J Invest Dermatol. 1997; 109: 25Abstract Full Text PDF PubMed Scopus (70) Google Scholar). Therapeutic intervention to prevent immune suppression by blocking one or more of these pathways may be beneficial in reducing the risk of skin cancer. An understanding of the immunoregulatory mechanisms can be aided by the use of agents that block the pathway at discrete steps, e.g., minimizing the effects of DNA damage by accelerating its repair, by the use of antioxidants to prevent cellular injury, and by using antibodies or other agents to block the action of immunosuppressive cytokines. Extracts of Aloe barbadensis are widely used as therapeutic agents for the treatment of minor cutaneous injuries. We have recently found that topical application of crude Aloe barbadensis gel prevents UV induced suppression of both local and systemic CHS and DTH immune responses (Strickland et al., 1994Strickland F.M. Pelley R.P. Kripke M.L. Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.J Invest Dermatol. 1994; 102: 197Abstract Full Text PDF PubMed Google Scholar). In this report, we examine some mechanisms by which crude and partially purified Aloe extracts prevent UV induced immunosuppression. Specific-pathogen-free female C3H/HeN Cr (MTV–) mice were purchased from the Animal Production Area of the Frederick Cancer Research Facility (Frederick, MD) and were maintained in a pathogen-free barrier facility in accordance with the National Institutes of Health and the American Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. The mice were housed in filter protected cages and provided with National Institutes of Health open formula mouse chow and sterile water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee. Each experiment was performed with aged matched mice that were 10–12 wk old. The Aloe barbadensis gel preparations used were "Aloe Research Foundation Standard Gel Samples" prepared for the Aloe Research Foundation by AloeCorp (Harlingen, TX), as previously described (Strickland et al., 1994Strickland F.M. Pelley R.P. Kripke M.L. Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.J Invest Dermatol. 1994; 102: 197Abstract Full Text PDF PubMed Google Scholar). These "Process A" materials were from lots ARF'91 A, ARF'94B, ARF'94G, and ARF'94K (Pelley et al., 1993Pelley R.R. Wang Y.-T. Waller T.A. Current status of quality control of Aloe barbadensis extracts.SOFWJ. 1993; 119: 255Google Scholar). In general, the composition of matter of these lyophilized materials was 9.6% polysaccharides, 11% glucose, 27% divalent metal cations and multivalent organic acids, and less than 5% materials extractable with organic solvents. The remainder consisted of univalent metal cations, chloride, and univalent organic acids. At the time of lyophilization, the total bacterial content was always under 30,000 per ml of gel. Although this extract was prepared on an industrial scale using commercial equipment, it does not correspond to any commercial product. An oligosaccharide-rich material was prepared from crude "Process A" gel by activation with cellulase followed by separation of small molecules from enzyme and polysaccharide by ultrafiltration. Frozen ARF 94K crude Aloe gel (2 liters, solids content 14 g) was thawed and 2.3 mg of crude commercial cellulase (Cellulase 4000, Valley Research, South Bend, IN) was added. The enzyme–Aloe mixture was ultrafilter fractionated by multiple cycles through a 5000 dalton cut-off polysulfone hollow fiber apparatus (2790 cm2, A/G Technology, Needham, MA). Each cycle consisted of passing the material, at ambient temperature, through the ultrafilter at a pressure sufficient to produce diffusate at a rate of 2 liters per h. Retentate volume was kept constant by the addition of deionized water. As each 2 liter portion of diffusate was produced, it was removed and concentrated by lyophilization. Four cycles of enzymatic treatment–ultrafiltration were performed. The first diffusate yielded 60% of the total dialyzable mass, the second yielded 24%, and the third and fourth diffusates combined accounted for 12% of the total dialyzable mass. More highly purified oligosaccharides were prepared by cleavage of purified Aloe polysaccharides with partially purified cellulase followed by separation of oligosaccharides from enzyme and polysaccharide by alcohol precipitation. Polysaccharide was purified from lyophilized "Process A"Aloe, lot ARF94K by modification of the method of Gowda using exhaustive dialysis followed by precipitation at 80% vol/vol with absolute ethanol at 4°C (Gowda et al., 1979Gowda D.C. Neelisiddaiah B. Anjaneyalu Y.V. Structural studies of polysaccharides from Aloe vera.Carb Res. 1979; 72: 201Crossref Scopus (69) Google Scholar). This yielded (14.2% of mass) a polysaccharide of expected sugar composition (7% glucose, 85% mannose, 4% galactose), almost all of which was in excess of 2,000,000 Da molecular weight. Cellulase was purified from crude T. reesei concentrated culture supernatants (lot ZPED, gift of Valley Research, South Bend, IN) by ethanol precipitation (50–80% fraction) and gel filtration upon Biogel P-200 (Bio-Rad, Richmond, CA). The protein concentration of the partially purified cellulase was determined by the Coomassie Blue dye binding assay (Bio-Rad, Richmond, CA). Oligosaccharide was produced by incubating 400 mg of purified polysaccharide with 12 μg of partially purified cellulase in 5 mM citrate buffer (pH 6) at ambient temperature for 2 h. This treatment reduced the viscosity of the solution by 50% but resulted in only a minor shift in the molecular weight distribution of the Aloe polysaccharides. Oligosaccharides were separated from precursor polysaccharide and enzyme by addition of absolute ethanol to 80% vol/vol and chilling to 4°C. The oligosaccharide containing supernatant was then separated from the precipitate (which contained the enzyme and polysaccharide) by centrifugation. Oligosaccharide, measured as hexose, constituted only 1.5% of the mass of the supernatant after stripping and lyophilization (the vast bulk of the supernatant consisting of the sodium citrate buffer). Oligosaccharides used for in vitro culture were diluted in serum-free minimal essential medium (MEM; Gibco, Grand Island, NY) and filter sterilized through a 0.22 μm membrane. UV radiation was administered in vivo using a bank of six unfiltered FS40 sunlamps (National Biological, Twinsburg, OH). Approximately 65% of the energy emitted from these lamps is within the UVB range (280–320 nm) and the peak emission is at 313 nm. The average irradiance of the source was ≈4.5 W per m2 at 20 cm distance, as measured by an IL700 radiometer with an SEE280 filter and a W quartz diffuser (International Light, Newburyport, MA). A single FS-40 bulb was used to irradiate cultured keratinocytes. The output of the lamp was 4.7 J per m2 per s, at a tube-to-target distance of 23 cm. The spontaneously transformed murine keratinocyte cell line, Pam 212, was obtained from Dr. Stuart Yuspa (National Cancer Institute, Bethesda, MD). The cells were maintained in our laboratory in MEM supplemented with 10% fetal bovine serum from Gibco at 37°C in a 5% CO2 balanced air humidified atmosphere as previously described (Ullrich, 1994Ullrich S.E. Mechanisms involved in the systemic suppression of antigen-presenting cell function by UV irradiation: keratinocyte-derived IL-10 modulate antigen-presenting cell function of splenic adherent cells.J Immunol. 1994; 152: 3410PubMed Google Scholar). To determine the effects of UV and Aloe extracts on cytokine production, keratinocytes were removed from subconfluent cultures by treatment with 0.25% trypsin (Gibco) and plated into sterile 100 mm plastic tissue culture-grade petri dishes (Corning Glass Works, Corning, NY) at a cell density of 2.5 × 106 cells per dish, and incubated in MEM/10% fetal bovine serum overnight at 37°C. The monolayers were washed three times with phosphate buffered saline (PBS), overlaid with 5 ml of PBS, and exposed to 300 J per m2 of UVB radiation. The cells were washed with PBS, overlaid with a solution of filter sterilized Aloe oligosaccharides in serum-free medium at 37°C. Endotoxin contamination of the Aloe was measured using the Limulus amebocyte lysate assay (Cape Cod Associates, Woods Hole, MA) and was found to be below the limit of detection (0.125 ng per ml). After 1 h the supernatant was removed, the cells were washed three times with PBS, and the incubation was continued for 24 h in serum-free medium. Control cultures were identically treated but not exposed to UV radiation. The supernatants were collected and the protein concentration was determined by the Coomassie Blue dye-binding assay using a bovine serum albumin standard. For cytokine quantitation, the culture supernatants were concentrated using the centriprep concentrating system (Amicon, Beverly, MA) and stored at –20°C. Groups of five mice were anesthetized with Nembutal (sodium pentobarbital, 0.01 ml per g body weight) ip and their shaved ventral skin was exposed to a single dose of 2 kJ UVB radiation per m2. Within 5 min of UV irradiation, the UV exposed skin was treated with Aloe extract in PBS or a control polysaccharide, methylcellulose (Sigma, St. Louis, MO) in PBS. Control animals were treated in an identical manner but were not exposed to UV radiation. Three days later the mice were sensitized on their shaved abdominal skin by applying 400 μl of 0.5% fluoroscein isothiocyanate (Molecular Probes, Eugene, OR) in acetone:dibutylphthalate (1:1 vol:vol). Five days after sensitization, the mice were challenged by applying 5 μl of 0.5% fluoroscein isothiocyanate on both the dorsal and the ventral surfaces of each ear. In some experiments, animals were sensitized with 0.3% 2,4-dinitrofluorobenzene (DNFB; Aldrich, Milwaukee, WI) in acetone. In those experiments, the mice were challenged with 0.2% DNFB applied to the ears as described above. Control mice were challenged with the hapten but were not sensitized. Ear thickness was measured using an engineers' micrometer (Production Tools, Houston, TX) immediately before challenge and 24 h later. Systemic suppression of the DTH response was induced using a single exposure to UVB radiation as follows. The dorsal fur of the mice was shaved with electric clippers, the animals were put into cages with plexiglas dividers, one mouse per chamber, and the cage covered with a wire lid. The incident light received by the animals under these conditions was reduced to 2.6 W per m2, by the shielding from the wire cage top. The animals were given a 5 kJ per m2 dose of UVB radiation in a single exposure. Within 5 min of UV irradiation, the UV exposed skin was treated with Aloe extract in PBS or a control polysaccharide, methylcellulose (Sigma) in PBS. Three days later, the mice were injected subcutaneously in each flank with 1 × 107 formalin fixed C. albicans cells. Ten days after sensitization, the mice were challenged with 50 μl of commercially prepared soluble Candida antigen, supplied as a 1:100 dilution (ALK Laboratories, Wallingford, CT) in each hind footpad. Footpad thickness (dorsal to plantar aspect) was measured immediately before challenge and 24 h later. Control mice were not sensitized with yeast cells but were challenged in both hind footpads with the Candida antigen. Specific footpad swelling was determined by subtracting the average values obtained from mice challenged but not sensitized. The percentage restoration of immunity in UV irradiated animals treated with oligosaccharides was calculated using the following formula:μmswellingunirradiatedAloetreated−μmswellingUVirradiatedAloetreatedμmswellingunirradiatedAloetreated×100% The response of UV irradiated, untreated mice was set as 0% restoration, whereas values for unirradiated, Aloe treated groups were considered as 100% response. Groups of five C3H/HeN mice were injected intravenously with Pam 212 keratinocyte supernatants. Each animal received 0.5 ml of supernatant containing 15–20 μg of protein. Three days later, the mice were immunized by subcutaneous injection of 107 formalin fixed C. albicans cells and their DTH response was elicited 10 d after sensitization using 50 μl of Candida antigen in each hind footpad. Murine IL-10 and IL-4 were measured using commercial kits or kit reagents purchased from PharMingen (San Diego, CA). Murine IL-13 was quantitated using a kit purchased from R&D Systems (Minneapolis, MN). All analyses were performed according to the manufacturers' instructions. The optical density was measured using a MR 5000 microplate reader (Dynatech, Chantilly, VA). Dorsal skin of C3H/HeN mice was shaved with electric clippers and exposed to 15 kJ UVB radiation per m2, followed immediately by treatment with a solution of Aloe extract in PBS. Control mice were similarly treated but were not exposed to UV radiation. Four days later, the mice were killed and the remaining fur in the treatment area was removed with a razor blade. The skin was excised, subcutaneous fat and connective tissue was removed, and the samples were frozen in liquid nitrogen. Cryostat sections were fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Ft Washington, PA) in PBS and stained as previously described (Nishigori et al., 1996Nishigori C. Yarosh D.B. Ullrich S.E. Vink A.A. Bucana C.D. Roza L. Kripke M.L. Evidence that DNA damage triggers interleukin 10 cytokine production in UV irradiated murine keratinocytes.Proc Natl Acad Sci USA. 1996; 93: 10354Crossref PubMed Scopus (215) Google Scholar). Pam 212 keratinocytes, cultured on 18×18 mm glass coverslips (Fisher Scientific, Pittsburgh, PA), were exposed to 300 J UVB per m2, and treated as described above. Twenty-four hours later the cells were fixed with 2% paraformaldehyde and stained for IL-10 protein. The cells were counterstained with one drop of Gill's hematoxylin for 30 s, rinsed with distilled water until the wash was clear, and mounted in universal mount (Research Genetics, Huntsville, AL). Microscopic images were recorded by digitizing the images using a 3CCD color camera (Sony, Tokyo, Japan), 24 bit True color frame grabber, and imaging system (Meyer Instruments, Houston, TX). The images were stored on 65 MB optical disks (Pinnacle Micro, Irving, CA) and analyzed using Optimas image-analysis software (Optimas, Bothell, WA). Images were printed using a digital printer (Sony). Statistical analyses were performed using ANOVA or two tailed Student's t test. A p value of < 0.05 was considered significant. Analyses were performed using Statview SE + Graphics software (Abacus Concepts, Berkeley, CA) on a Macintosh LC II microcomputer. We have previously shown that cutaneous application of a crude extract of Aloe barbadensis gel protects both CHS and DTH responses in mice from suppression by UV radiation (Strickland et al., 1994Strickland F.M. Pelley R.P. Kripke M.L. Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.J Invest Dermatol. 1994; 102: 197Abstract Full Text PDF PubMed Google Scholar). This study investigates the mechanism of immune protection by first determining whether protection of CHS and DTH responses are mediated by the same or different agents in Aloe gel. Protection against UV induced local immune suppression of CHS was measured by exposing the shaved ventral skin of C3H/HeN mice to 2 kJ UVB radiation per m2 and applying a solution of 5 mg Aloe gel extract per ml (lot ARF91 A, "Process A") in PBS to the irradiated skin immediately after exposure. The animals were sensitized 3 d later by applying hapten through their ventral skin. Unirradiated control groups were treated with the Aloe gel and sensitized. The data obtained from three lots of Aloe gel (ARF91 A, ARF94B, and ARF94G) were combined and expresssed as the mean ± SEM percentage unirradiated matching control. The responses to antigen varied between experiments; however, typical positive control values for DTH responses to C. albicans ranged from 200 to 300 μm footpad swelling above background (Strickland et al., 1994Strickland F.M. Pelley R.P. Kripke M.L. Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.J Invest Dermatol. 1994; 102: 197Abstract Full Text PDF PubMed Google Scholar; not shown). Responses to hapten ranged from 70 to 150 μm ear swelling for fluoroscein isothiocyanate and 150–180 μm for DNFB. In groups of UV irradiated mice treated with PBS alone, a 50–80% reduction in CHS and DTH responses was observed compared with their unexposed, matching controls. This level of response was considered as 0% restoration whereas the response of unirradiated, Aloe treated matching controls was set as 100%. The results presented in Figure 1 show that treatment of UV irradiated skin with the Aloe extract partially prevented suppression of the CHS response to hapten. The protection afforded by the three different lots of gel varied. For example, 3 mo after manufacture, application of ARF91G gel to UV irradiated animals provided only 43% restoration of their CHS response, whereas the ARF91A lot of gel extract completely restored the CHS response. The activity of ARF91B was intermediate between these two values. The levels of protection provided by the gel were maximal at a dose of 5 mg per ml (wt:vol) and could not be improved by increasing the dose of Aloe used (not shown). The activity of all three lots of Aloe gel extract decayed with time, despite their storage as lyophilized powder. After 9 mo, none of the extracts prevented UV induced suppression of CHS responses to hapten Figure 1. Other lots of Aloe gel "Process A" extract gave similar results except that the levels of CHS protection ranged from 30 to 100% and longevity of CHS protective activity ranged from 3 to 9 mo post-manufacture. Commercially prepared (non-"Process A") Aloe gels from the same source were uniformly inactive even when tested within 1 mo of manufacture. Systemic suppression of DTH responses was measured in mice whose shaved dorsal skin was exposed to 5 kJ UVB per m2, treated with the same lot of gel extract used for CHS protection studies, and sensitized by a subcutaneous injection of C. albicans 3 d later. In contrast to the partial protection of the CHS responses to hapten, Aloe gel completely prevented systemic suppression of DTH to C. albicans. The immunoprotective activity of all three lots of lyophilized extract remained unchanged after 12 mo of storage Figure 1. At each time point at which the Aloe gel was tested, the experiment was repeated at least once in order to confirm the results. Restoration of immunity was not due to nonspecific immunostimulation by the Aloe gel, because neither CHS nor DTH responses were affected in unirra