| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; and
AGI Dermatics, Freeport, NY 11520
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously demonstrated that reducing the number of cyclobutane pyrimidine dimers (CPD) in UV-irradiated skin abrogated the systemic suppression of contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) to Candida albicans (13, 14). From these results, we inferred that unrepaired DNA damage stimulates keratinocytes to produce cytokines, thereby modifying critical steps in the immunologic pathway and ultimately leading to reduced cell-mediated immune responses and generation of suppressor T cells (Ts) to certain Ags. Other reports support our hypothesis. Hurks et al. (15) reported that the action spectra for UV-induced suppression of the mixed lymphocyte and mixed epidermal cell-lymphocyte reactions closely resemble those for the induction of CPD and 6-4 photoproducts. Action spectra for local and systemic suppression of CHS are also consistent with DNA damage as an initiator of these effects (16, 17). Also, XPA knockout mice, which are deficient in DNA repair, were reported to show increased local and systemic suppression of CHS responses after UV irradiation compared with wild-type mice (18). Recently, we provided the first direct evidence that UV-induced DNA damage in the form of CPD initiates cytokine production, providing further support for the hypothesis that UV-induced DNA damage triggers the production of immunomodulatory epidermal cytokines (19).
Having demonstrated that DNA damage is a primary molecular mechanism for initiation of immune suppression in these models, we hypothesized that DNA damage other than CPD might also cause immune suppression. To test this hypothesis, we used the HindIII restriction enzyme, which recognizes the 5'-AAGCTT-3' base sequence in dsDNA and causes double-strand breaks at these sites. This restriction endonuclease is widely used and well characterized and does not directly induce membrane damage. This 6-base cutter averages about 5000 bases between double-strand breaks in purified DNA. Recently, we showed that HindIII encapsulated in liposomes induces both local and systemic suppression of CHS in vivo (20). In this study, we extended these findings by investigating whether damage caused by the restriction enzyme also suppressed the DTH responses to C. albicans and alloantigen and induced Ts induction; we also examined the effect of HindIII liposomes on the immunosuppressive activity of keratinocyte culture supernatants and on IL-10 production in vivo and in vitro.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Specific pathogen-free C3H/HeNCrMTV- and BALB/cAnNCr female mice were obtained from the Frederick Cancer Research Facility Animal Production Area (Frederick, MD). Age-matched mice between 10 and 12 wk of age were housed in filter-protected cages, and ambient lighting was controlled to provide 12-h light/12-h dark cycles. Autoclaved National Institutes of Health open formula mouse chow and water were provided ad libitum. The animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International; procedures were approved by the institutional animal care and use committee.
HindIII endonuclease in liposomes
HindIII restriction enzyme (New England Biolabs,
Beverly, MA) was purified as previously described (20). The enzyme was
diluted to 10,000 U/ml in the storage buffer (250 mM NaCl, 10 mM
Tris-Cl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, and 500 g/ml BSA) and in some
cases inactivated by heating at 65°C for 60 min. The enzyme was
filtered through a 0.2-µm SFCA filter (Nalgene Division, Sybron,
Rochester, NY) and encapsulated in pH-sensitive liposomes, as described
previously (20). These liposomes are taken up by cells in culture and
pass through the stratum corneum into the epidermal cells of mice,
where they release endonucleases intracellularly (21). The encapsulated
HindIII activity was 400 U/ml, as assayed by the
manufacturer’s directions, using DNA as substrate and adding 0.1%
Triton X-100 to dissolve the liposomes. Control liposomes contained
either T4 endonuclease V (T4N5 liposomes, previously described by
Kripke et al. (14)) or heat-inactivated HindIII. Empty
liposomes were prepared identically, except that proteins were omitted.
For in vivo use, the liposomes were mixed into 1.5% neutralized Hypan
SS201 hydrogel (Lipo Chemicals, Paterson, NJ) in PBS, pH 8, to a final
concentration of 40 U/ml and applied to shaved mouse skin (
10
U/mouse) with a moist cotton swab. For in vitro use, the liposome
preparation was diluted with MEM (Life Technologies, Grand Island, NY)
supplemented with 1% FCS (Life Technologies) to a final concentration
of 12 U/ml, followed by filtration through a 0.22-µm filter. Cells
were treated with 3 ml of liposomes/100-mm dish (for collecting
supernatant) and 1.3 ml/35-mm dish (for immunohistochemistry) for
1 h at 37°C.
Detection of DNA damage
Skin was excised from the mice 4 h after the HindIII liposome treatment, and epidermis was separated after the skin was treated with 1.5 U/ml dipase in PBS (Boehringer Mannheim, Indianapolis, IN). DNA was extracted from the epidermis, followed by electrophoresis in a 4% neutral agarose gel. Negatives of the photograph were scanned, and the frequency of double-strand breaks was analyzed as previously described (20). An excess of 13 to 20 double-strand breaks per DNA megabase pair was produced by HindIII liposome treatment (not shown), similar to our previous report (20).
Immunohistochemical analysis for IL-10 and p53
IL-10 in the skin and in Pam212 cells was examined by an immunoperoxidase method using monoclonal rat anti-mouse IL-10 (clone JES5-2A5, American Type Culture Collection, Rockville, MD). p53 was examined using mouse polyclonal anti-human p53 Ab Ab-1 (clone 421, Oncogene Science, Uniondale, NY). HindIII liposome-treated and UV-irradiated mouse skin was collected 1, 3, 5, 8, and 12 days after treatment, embedded in OTC compound, and frozen in liquid nitrogen; 4-µm cryostat sections were fixed with 2% paraformaldehyde. The slides were placed in a humidified chamber, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide; slides were incubated with protein blocking solution (10% normal horse serum and 1% normal goat serum) for 20 min, followed by treatment with rat anti-mouse IL-10 mAb overnight at 4°C. The slides were washed with PBS and incubated for 30 min with biotin-labeled rabbit anti-rat Ab (Boehringer Mannheim), for 20 min with peroxidase-labeled streptavidin, and for 20 min with diaminobenzidine (Research Genetics, Huntsville, AL). For p53 staining, skin sections were fixed with 2% paraformaldehyde at room temperature for 10 min, followed by methanol at -20°C for 10 min. Endogenous peroxidase activity was again blocked with 3% hydrogen peroxide, and sections were incubated with protein blocking solution for 20 min at 25°C and with Ab-1 Ab (clone 421, Oncogene Science) overnight at 4°C, followed by peroxidase-labeled rat anti-mouse IgG2a. Other procedures were the same as described for IL-10 staining.
For cultured cells, Pam212 cells were grown on coverslips in 35-mm dishes. On the following day, cells were treated with 1.3 ml of HindIII liposome solution under the same conditions as those used for supernatant harvest. Twelve and twenty-four hours after UV irradiation, cells were washed with PBS and fixed with 2% paraformaldehyde for 10 min at 25°C; they were rinsed with PBS and stained by the method used for skin sections, except that endogenous peroxidase blocking was omitted.
UV source
The UV source for irradiation of mice was a bank of six FS40 sunlamps (National Biologic, Twinsburg, OH), which emit about 65% of their radiation within the UV-B (280–320 nm) range and have a peak emission at 313 nm. The average irradiance of the source was about 9 watt/m2, as measured with an IL-1700 radiometer and an SEE 240 UV-B detector equipped with an A127 quartz diffuser (International Light, Newburyport, MA). The dose rate of the incident radiation received by the animals was decreased to 4.5 watt/m2 by the cage lid screening. Before irradiation, the dorsal fur of the mice was shaved with electric clippers, and the animals were placed in individual compartments in cages located 20 cm below the lights.
A single FS40 sunlamp was used to irradiate keratinocytes. The irradiance of the source averaged 1.43 watt/m2 at a tube-to-target distance of 23 cm.
DTH responses to Candida and alloantigen
Groups of five C3H mice were sensitized by injecting 0.2 ml of 1 x 107 formalin-fixed C. albicans cells (14) or 2.5 x 107 allogeneic BALB/c spleen cells s.c. into each flank. Nine (Candida) or six (alloantigen) days later, the hind footpad thickness was measured with a spring-loaded micrometer (Mitutoyo, Tokyo, Japan), and the mice were challenged by intradermal injection of 50 µl of Candida Ag (Alerchek, Portland, ME) or 1 x 107 BALB/c spleen cells in both hind footpads. Footpad thickness was measured again 24 h later, and the swelling was determined by subtracting prechallenge from postchallenge measurements. Negative controls consisted of mice that were challenged but not sensitized.
Test for Ts activity
Spleens from mice treated with HindIII liposomes or UV-B and sensitized with C. albicans were removed 10 days after sensitization. Single-cell suspensions were prepared from pooled spleens in HBSS. Clumps were removed by filtration through nylon mesh. The cells were washed once and refiltered, and 1 x 108 viable, nucleated cells were injected i.v. into groups of five syngeneic recipients. Immediately thereafter, the recipients were sensitized with C. albicans; the DTH response was measured 9 days later as described above.
Neutralization of IL-10 in vivo
Groups of five C3H mice were treated with HindIII liposomes. Four and twenty-four hours later, they were injected i.p. with either 100 µg of monoclonal rat anti-mouse IL-10 (purified from JES5–2A5 supernatants by use of protein A/G columns; Pierce, Rockville, IL) or 100 µg of IgG1 from normal rat serum. The mice were immunized with Candida, and the DTH response was measured as described above.
Cell line
A spontaneously transformed, BALB/c keratinocyte cell line, Pam212, obtained originally from Dr. Stuart Yuspa, National Cancer Institute (Bethesda, MD), was used. Cells were maintained in MEM supplemented with 10% FBS, sodium bicarbonate (0.075%), L-glutamine (2 mM), and nonessential amino acids. Cells were incubated at 37°C in 95% air/5% CO2.
Supernatants from HindIII liposome-treated keratinocytes
A total of 4 x 106 Pam212 cells were plated in 100-mm tissue culture dishes in 8 ml of medium. On the following day, the medium was removed, and cells were washed twice with PBS and treated with HindIII liposomes (12 U/ml) in MEM containing 1% FCS at 37°C for 1 h, after which the cells were washed twice with PBS and cultured in serum-free medium. Twenty-four hours later, the supernatants were harvested. The protein concentration of the supernatants was determined using bicinchoninic acid (BCA protein assay reagent, Pierce), and supernatants were injected i.v. into mice.
C3H mice were injected with 20 to 25 µg of supernatant protein from HindIII liposome-treated keratinocytes. Control supernatants were prepared by treating keratinocytes with control liposomes or UV-B radiation. Five days later, the mice were immunized with allogeneic BALB/c spleen cells and challenged as described above. The interval between supernatant injection and immunization was selected to mimic the protocol used in studies by Rivas and Ullrich (11), which was based on the optimal time of immune suppression following UV irradiation in vivo.
Determination of IL-10 by ELISA
IL-10 release from Pam212 cells was measured by ELISA using the same supernatants that were injected into mice. Rat anti-mouse IL-10 (JES5-2A5) was used as a capture Ab in combination with a biotinylated detection Ab (SXC-1, PharMingen, San Diego, CA). ELISA was conducted according to the manufacturer’s procedures, and the IL-10 concentration was determined from the linear portion of a standard curve obtained using rIL-10 (PharMingen).
Statistical analyses
All experiments were performed at least twice and usually three times; results shown are representative of all experiments. Groups of five mice were used for DTH assays, and the significance of differences was determined using Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Application of HindIII liposomes to murine
skin produced double-strand breaks in epidermal DNA, as measured by
electrophoresis on neutral agarose gels (18). With the liposome
preparation used in these studies, an excess of between 13 and 20
double-strand breaks per DNA megabase pair was produced (not shown).
Because p53 protein accumulates in cells in response to DNA damage
(22), we examined the p53 content of cells in the skin by
immunoperoxidase staining as an additional marker of the in vivo
activity of HindIII liposomes. This approach also
demonstrated the ability of HindIII liposomes to produce DNA
damage in vivo, since application of the HindIII liposome
preparation to the skin of mice increased the amount of p53 in
epidermal cells (Fig. 1
). One day after HindIII liposome
treatment, p53 was present in the epidermal cells, almost to the same
extent as after UV irradiation (Fig. 1, A and B).
By day 3, some p53+ cells remained in the basal layer of
the epidermis of UV-irradiated mice (Fig. 1C), but in
HindIII liposome-treated skin (Fig. 1D), p53 staining had returned to background levels (Fig. 1E). There was also considerably more hyperplasia in
UV-irradiated skin than in HindIII liposome-treated skin
(Fig. 1, C vs D). Liposomes containing
heat-inactivated HindIII induced no detectable increase in
p53 (not shown).
|
To test whether DNA-damaging agents other than UV-B could also
suppress DTH responses, we applied HindIII liposomes to
mouse skin and immunized the animals 5 days later with alloantigen or
C. albicans. Treatment with HindIII liposomes
reduced the DTH response to alloantigen to the same extent as UV-B,
whereas treatment with control liposomes containing T4 endonuclease V
(T4N5 liposomes), an enzyme involved in excision repair of CPD, had no
effect on the DTH response in the absence of UV irradiation (Fig. 2). Similarly, the DTH response to
C. albicans was reduced (Fig. 3A). In contrast to the effect
of UV-B radiation, however, transfer of spleen cells from mice treated
with HindIII liposomes failed to impair the induction of DTH
to C. albicans, suggesting that Ts had not been induced by
this treatment (Fig. 3B).
|
|
IL-10 has been reported to play an essential role in UV-induced
suppression of DTH responses (11, 23). We asked, therefore, whether
IL-10 also plays a role in suppression of DTH by HindIII
liposome treatment. A neutralizing anti-IL-10 mAb was injected 4
and 24 h after HindIII liposome treatment, and the mice
were sensitized on day 5 after liposome treatment. As shown in Figure 4, injection of anti-IL-10 Ab
prevented the suppression of DTH in response to C. albicans,
whereas an isotype-matched control Ab had no effect.
|
Previous studies demonstrated that soluble factors secreted by
UV-B-irradiated murine keratinocytes can suppress immune responses in
vivo (9, 24); one such factor is IL-10 (11, 19, 23). We therefore
determined whether HindIII liposomes would induce such
immunosuppressive activity in vitro also. Cultures of Pam212 cells were
treated with HindIII liposomes, UV-B (positive control), or
T4N5 liposomes (negative control), and supernatants were harvested
24 h later. The supernatants were injected i.v., and 5 days later
the mice were immunized with alloantigen, and the DTH response was
measured after an additional 6 days. Supernatants from keratinocyte
cultures treated with HindIII liposomes inhibited the DTH
response, as did those from UV-B-irradiated cells, whereas those from
cells treated with T4N5 liposomes had no effect on the DTH response
(Fig. 5A). Spleen cells from
these groups of mice were transferred to naive mice, which were then
immunized with allogeneic spleen cells to test for Ts activity. As
shown in Figure 5B, spleen cells from mice receiving
supernatants from UV-B-treated keratinocytes contained Ts activity;
however, those from mice receiving supernatants from cells treated with
HindIII liposomes or T4N5 liposomes had no Ts activity.
|
The ability of HindIII liposomes to induce IL-10
production in mouse skin in vivo was analyzed by mAb and
immunoperoxidase staining of skin sections. IL-10 could be detected in
the skin of mice treated with HindIII liposomes (Fig. 6, B, D, and
F) as well as with UV-B (Fig. 6, A, C,
and E). Staining was apparent on days 1, 3, and 8 after
treatment, but both the intensity of staining and the number of
positive cells were greater in the UV-B-irradiated skin than in the
HindIII-liposome-treated skin. Occasional IL-10+
dendritic cells were evident in the dermis of UV-B-irradiated mice but
not in that of HindIII-liposome-treated mice. Neither skin
treated with heat-inactivated HindIII in liposomes (Fig. 6G) nor untreated skin (Fig. 6H) showed
detectable staining with anti-IL-10 Ab.
|
shows the results of immunoperoxidase
staining for IL-10 in Pam212 cells 12 h (Fig. 7C) and
24 h (Fig. 7D) after treatment with HindIII
in liposomes; untreated cells (Fig. 7, A and B)
and cells treated with heat-inactivated HindIII liposomes
(Fig. 7, E and F) were only rarely positive for
IL-10 staining. IL-10 was also increased in the supernatants of
cultures of Pam212 cells collected 24 h after treatment with
HindIII liposomes (940 pg/ml) and UV-B radiation (550
pg/ml), compared with those from cells treated with T4N5 liposomes
(<40 pg/ml) or untreated cells (40 pg/ml).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B and
activating protein-1 (25, 26), photoisomerization of UCA (12),
and generation of H2O2 (27). Using the
approaches of direct photoreactivation of CPD (7, 13) and excision
repair of CPD by T4N5 liposomes (14, 19), we have provided additional
evidence that DNA damage in the form of CPD initiates both local and
systemic suppression of CHS and systemic suppression of DTH to C.
albicans. As an additional test of the role of cutaneous DNA
damage as an initiator of immune suppression, we used
liposome-encapsulated HindIII restriction endonuclease to
induce double-strand breaks in DNA, an approach unlikely to generate
H2O2, membrane damage, or cis-UCA.
In earlier studies, we demonstrated that treatment of mouse skin in
vivo with these liposomes suppressed the induction of CHS to haptens
applied to either the treated or a distant, untreated site; however,
this treatment did not produce tolerance or suppression transferable
with spleen cells (20). These effects were due to the DNA cutting
ability of the HindIII enzyme, since heat-inactivated
HindIII in these same liposomes had no effect. On the other hand, studies using a mAb directed against cis-UCA suggested that UV-induced systemic suppression of DTH to alloantigen, sheep erythrocytes, and herpes simplex virus was mediated by cis-UCA and therefore was unlikely to involve DNA damage (28, 29). To test more critically the potential role of DNA damage in suppressing DTH responses, we extended our studies of liposomal HindIII-induced suppression of CHS responses to two models of DTH and an in vitro model for production of immunosuppressive factors, including IL-10. Based on previous studies, we predicted that HindIII liposomes would suppress DTH to C. albicans, which appears to depend on DNA damage (14), but not alloantigen, which appears to depend on cis-UCA (29). However, both DTH responses were inhibited by HindIII liposome treatment, and Ts could not be detected in either model. In addition, although treatment of keratinocytes in vitro with liposomal HindIII produced immunosuppressive activity and IL-10 in culture supernatants, these supernatants were unable to produce detectable Ts in recipient mice, in contrast to the effect of supernatants induced by UV-B irradiation. These results imply either that DNA damage per se does not account for all of the immunosuppressive properties of UV-B irradiation or that CPD are qualitatively different in their biologic consequences from double-strand breaks in DNA. Furthermore, they provide additional evidence that IL-10 alone is insufficient to reproduce all the immunomodulatory effects of UV-B radiation either in vivo or in vitro.
Previous work demonstrated that IL-10 has an important role in
suppressing DTH responses to UV-B irradiation (11, 23) and in inducing
tolerance to haptens applied to the site of UV-B irradiation (30).
Treatment of mice with mAb against IL-10 also restored
HindIII-induced suppression of DTH in response to
C. albicans, implying that IL-10 is involved in suppression
mediated by double-strand breaks in DNA as well. However, supernatants
from Pam212 cells treated with HindIII liposomes failed to
induce Ts, even though they contained as much IL-10 as those generated
by UV-B radiation. This result implies, as suggested by Rivas and
Ullrich (11), that production of IL-10 is necessary for immune
suppression by UV-B irradiation, but is insufficient to account for all
of its immunosuppressive properties, particularly Ts induction. This
contrasts with the CHS model, in which IL-10 appears to play a role in
tolerance induction (30) but not in primary immune suppression by
UV-B irradiation (31). In our in vivo studies with
HindIII liposomes, we demonstrated increased production
of IL-10 in the epidermis; however, it is clear from the
immunohistochemical staining for IL-10 that the amount and the timing
of IL-10 production in the epidermis and the IL-10 content of dendritic
cells in the dermis differed between mice treated with UV-B and
HindIII liposomes. Although these differences may account
for some of the discrepancies between the immunosuppressive effects of
UV-B and HindIII liposomes, it seems likely that factors
other than IL-10 are crucial contributors to Ts induction in the DTH
models. For example, less TNF-
is induced in murine keratinocytes by
HindIII liposomes than UV-B (20), and reactive
oxygen species may participate in some UV-B-induced immunosuppressive
effects (27).
These and our previous studies (20) demonstrate that HindIII
liposomes also increase the production of both IL-10 and TNF- in
vivo and in vitro. Unlike UV-B radiation, however, this treatment is
expected to produce DNA double-strand breaks without the formation
of reactive oxygen species, which can cause membrane damage and
additional types of DNA damage. These findings reinforce the idea that
direct DNA damage can trigger the production of immunomodulatory
cytokines, and they have important implications for the use of other
DNA-damaging agents, such as chemotherapeutic drugs, which are known to
cause leukopenia and immune suppression. Specifically, they raise the
possibility that the mechanism of action of such drugs may include
alterations in the regulation of cytokines that modify the production
or activity of hemopoietic cells as well as direct the cytotoxicity of
these cells and their precursors.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Margaret L. Kripke, Academic Programs, Box 147, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
3 Abbreviations used in this paper: UV-A, 320–400 nm UV radiation; PUVA, psoralen plus UV-A radiation; UV-B, 280–320 nm UV radiation; CPD, cyclobutane pyrimidine dimer(s); CHS, contact hypersensitivity; DTH, delayed-type hypersensitivity; Ts, suppressor T lymphocyte(s); T4N5 liposomes, liposomes containing T4 endonuclease V; UCA, urocanic acid.
Received for publication February 24, 1998. Accepted for publication May 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B (NFB) activity independently from chromosomal DNA damage in cell-free cytosolic extracts. J. Invest. Dermatol. 102:422.[Medline]
in the immune suppression induced by Ultraviolet radiation. J. Leukocyte Biol. 56:769.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  J. Jans, G. A. Garinis, W. Schul, A. van Oudenaren, M. Moorhouse, M. Smid, Y.-G. Sert, A. van der Velde, Y. Rijksen, F. R. de Gruijl, et al. Differential Role of Basal Keratinocytes in UV-Induced Immunosuppression and Skin Cancer Mol. Cell. Biol., November 15, 2006; 26(22): 8515 - 8526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Y. Crowe, J. M. Coquet, S. P. Berzins, K. Kyparissoudis, R. Keating, D. G. Pellicci, Y. Hayakawa, D. I. Godfrey, and M. J. Smyth Differential antitumor immunity mediated by NKT cell subsets in vivo J. Exp. Med., November 7, 2005; 202(9): 1279 - 1288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Walterscheid, S. E. Ullrich, and D. X. Nghiem Platelet-activating Factor, a Molecular Sensor for Cellular Damage, Activates Systemic Immune Suppression J. Exp. Med., January 14, 2002; 195(2): 171 - 179. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Miyauchi-Hashimoto, K. Kuwamoto, Y. Urade, K. Tanaka, and T. Horio Carcinogen-Induced Inflammation and Immunosuppression Are Enhanced in Xeroderma Pigmentosum Group A Model Mice Associated with Hyperproduction of Prostaglandin E2 J. Immunol., May 1, 2001; 166(9): 5782 - 5791. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Hill, V. K. Shreedhar, M. L. Kripke, and L. B. Owen-Schaub A Critical Role for Fas Ligand in the Active Suppression of Systemic Immune Responses by Ultraviolet Radiation J. Exp. Med., April 19, 1999; 189(8): 1285 - 1294. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
