Aug. 1982 DERMATOPHYTOSIS OF GUINEA PIG SKIN ON NUDE MICE 129

15. Rygaa rd J: S kin gra fts in nude mice. 2. Rat skin gra fts in nude mice of three gene tic backgrounds (BALB/c CH3, C57/ BL) . Acta Pa t h Microbiol Scand 82:93-104, 1974

16. Rygaard J : Skin gra fts in nude mice. 3. Fate of graft.s from man a nd donors of other taxonomic classes. Acta Path Microbiol Scand 82: 105-112, 1974

17. Kreider JW, Bartlett GL, Sharkey FE: Primary neoplastic trans­formation in vivo of xenogeneic skin grafts in nude mice. Cance r Res 39'273-276 1979

18. Reed ND , Manning DO: Present status of xenotransplantation of nonmalignant tissue to the nude mouse, Fogh J , Giovanella BC (eds) : The Nude Mouse in Experimental and Clinical Research, New York, Academic Press, Inc, 1978, pp 167- 185

19. I(rueger GG, Manning DO, Ma lone J, Ogden B: Long-term main­tenance of psoriatic human skin on congeni ta lly a thymic (nude) mice. J Invest Oennatol 64:307-312, 1975

20. Manning DO, Krueger GG: Use of cyanoacrylate cement in skin grafting congenita lly athymic (nude) mice. Tra nsplantation 18:380-383, 1974

21. Lillie RD , Fullmer HM: Histopathologic T echniqu e and Practical Histochemistry, 4th ed. New York, The Bla kis ton Divis ion, McG raw-Hili Book Co., 1965

22. Kligman AM: Tinea capitis due to M. A udouinii and M. callis. Arch Dermatol 71 :313-337, 1955

23. Sohnle PG, Frank MM , Kirkpatrick CH: Mecha nism invovled in elimination of organisms from expermenta l cutaneous Can dida. a llJ ica,ns infections in guinea pigs, J ImmunoII1 7:523- 530, 1976

24. KorsZLIn AK, Wilton JM , Johnson NW: The ill vivo effects of Iymphokines on mitotic activity a nd keratiniza t ion in gu inea pig epidermis. J Invest Dermatol 7G:433-437, 1981

25, Lav ia MF, Hill RB: Principles of Pathobiology, 2nd ed, London, Oxford University Press, 1975, p 205

0022-202X/82/7902·0 l29$02.00/ 0 TH E JO U HNAL OF INV ESTIG ATIVE D E HMATOLOGY, 79: 129- 135, 1982 Copyright © J982 by The Williams & Wilkins Co.

2G, Kligman AM: Pathophysiology of I'i ngworm infections in animals with skin cycles. J Invest Dermatol 27:171- 185, 1956

27, Eaton GJ: Ha ir growth cycles and wave pa tte rns in " nude" mice, Transplanta t ion 22:2 17-222, 1976

28, Drouchet E, Dompmartin D , Papachristou -Moraiti A, Ravis e P: Dermatite experimenta le a PityrosporlJlI/ ovale et (ou) Pi tyros­porum orbiculare chez Ie cobaye e t a l souris. Sabouradia 18: 149-156, 1980

29, Blank H, Sagami S, Boyd C, Ro th FJ : The pathogenesis of super­fi cial fungous infections in cul tured human skin. Arch Dermalol 79:524-535, 1959

30. Carlis le DH, Inouye J C, King RD, Jones H E: S ignificance of serum fungal inhibitory factor in dermatophytosis, J Invest Dermatol 63:239-24 1, 1974

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32. Green F, Balish E: Trichophyton mentagrophytes dermatophylosis in germ-free guinea pigs, J Invest D ermaLol 75:476-480, 1980

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VoL 79, No, 2 Printed ill U. ,A,

Langerhans Cells as Stimulator Cells in the Murine Primary Epidermal Cell-Lymphocyte Reaction: Alteration by UV-B Irradiation

WERNER ABERER, M.D., GEORG STINGL, M.D., LAURA A. STINGL-GAZZE, A.B., AND

KLAUS WOLFF, M.D.

First Department of Dennatology, University of Vienna M edica.l School, Vienna, A ustria

I region dependent functions of immunocompetent cells, Le., induction of antigen specific, allogeneic and syngeneic T cell activation, have been reported to be highly UV susceptible. Since ' the epidermis is the only tissue which is naturally exposed to UV-irradiation, we

M anuscript received October 18, 1981; accepted for publication J an­uary 28, 1982,

Reprint requests to: Dr, Werner Aberer, 1st D ept. of D ermatology, University of Vienna, Alsers trasse 4, A-1090 Wien, Austria.

Abbreviations: aELR: a llogeneic epidermal cell-lymphocyte reaction aMLR: allogeneic mixed leukocyte reaction C': complement EC: epidermal cell ELR: epidermal cell-lymphocyte reaction Ia antigen: immune response-associated antigen LC: epidermal Langerhans cell MHC: major histocompatibility complex mJ /cm2

: milli -Joules per squal'e centimeter MLR: mixed leukocyte l'eaction NMS: normal mouse serum sELR: syngeneic epidermal cell-lymphocyte reaction sMLR: syngeneic mixed leukocyte reaction UV: ultraviolet ligh t UV -B: ultraviolet B , 290-320 nm

conducted experiments to investigate whether murine epidermal cells (EC), particulal·ly la-positive Langer­hans cells (LC), could induce syngeneic and allogeneic T cell activation and, if so, whether these functions could be altered by UV-irradiation.

Epidermal cell-lymphocyte cultures (ELR) were estab­lished using either nonirradiated or irradiated (1-40 mJ/ cmz UV -B) Balb/c (H_2d

) EC as stimulator cells and either Balb/c or C57Bl/6 (H_2 b

) purified T lymphocytes as r e­sponders.

Balb/c EC not only induced a vigorous proliferative response in C57Bl/6 T cells (peak response: day 5-6) but also substantially stimulated syngeneic T cells (day 7-9). Pretreatment ofEC with specific anti-Ia serum plus com­plement abolished their stimulatory capacities which demonstrates that LC are of critical importance in these reactions. UV-B irradiation of EC resulted in a dose­dependent reduction of their ELR-stimulatory capaci­ties. UV-B doses which virtually abolished the ELR stim­ulatory capacity of LC did not affect EC viability and only marginally reduced the protein synthesizing capac­ity ofEC.

The finding that I region dependent LC functions are particularly UV-susceptible may provide a useful tool

130 ABERER ET AL

for the modulation of the afferent limb of the immune response.

The mixed leukocyte reaction (MLR) is the proliferative response of a T lymphocyte subpopulation to certain leukocytes bearing defmed immunogenetic specificities. Depending upon whether or not differences in haplotype exist between stimula­tor and responder cells, the terms allogeneic MLR (aMLR) or syngeneic MLR (sMLR) are used. Whereas the biological sig­nificance of the sMLR is still poorly understood and may reflect the self-recognition capacities of T lymphocytes [1], one may assume that the aMLR represents an in vitro correlate of the graft vs.-host response [2] and of the induction of allograft rejection [3].

While it is generally agreed that, at least in the murine aMLR, Ly1 + T cells constitute the relevant proliferating re­sponder population [4], some controversy exists as to the exact nature of the stimulator cell population. Some authors assume that T or B lymphocytes have strong MLR-stimulatory capac­ities [5,6]; other investigators maintain that certain dendritic cells [7] represent the relevant stimulator cells in both the murine sMLR and aMLR. However, there is general agreement that the presence of cells expressing immune-response-associ­ated (la) antigens is essential for the elicitation of T cell activation in both sMLR and aMLR [8]. Although the aMLR can be elicited by antigens encoded for by the KID or I regions of the major histocompatibility complex (MHC), the aMLR stimulation induced by only K or D differences also depends upon stimulator cells expressing la antigens [9].

Several investigators have shown that ultraviolet light (UV)­irradiation of lymphoid cells makes them incapable of serving as MLR stimulators [10-12] but the mechanism by which UV interferes with MLR stimulator cell function is only poorly understood. Whereas some authors believe that UV influences the expression of MLR stimulatory determinants on stimulator cells [13], other investigators have shown that certain accessory cells critically needed for the generation of cytotoxic T lympho­cytes [14,15] or in vitro T cell dependent antibody responses [16] are particularly UV -susceptible and have implied that the UV-induced defect lies in the antigen-presenting cell.

The epidermis is the prime target tissue for UV. In mammals, it harbors a uniform population of bone-marrow-derived den­dritic, la-positive cells, termed Langerhans cells (LC) which have been shown to exhibit antigen-presenting function [17-19] and to be J>otent stimulators in the primary and secondary MLR [17,1!:S].

We demonstrate in this study that among murine epidermal cells (EC) only la-positive LC are capable of inducing syngeneic and allogeneic T cell proliferation and that this functional capacity is highly susceptible to UV.

MATERIALS AND METHODS Animals

Balb/c (H-2"), C57Bl/6 (H_2b) and NZB (H-2") mice were bred in

our own colony. The Balb/c and C57Bl/6 colony was initiated from animals obtained from the Zentralanstalt fur Versuchstiere, Hannover, FRG, the NZB mice from Olac Ltd., U.K. In all experiments male mice 10 to 15 weeks old were used.

Prf}paration of cell suspensions

Epidermal cells (EC) : Single EC suspensions from Balb/c ear skin were prepared by floating the split ears dermal-side-down on a 1% trypsin (GIBCO, Grand Island, N.Y.)-phosphate buffered salin~ (PBS) solu tion for 45 min at 37°C, removing the loosened dermis, treating the epidermis with a solution of 0.025% deoxyribonuclease (DNase 1, Worthington Labs., N.J.) in PBS and teasing it with the tip of a glass stirring rod. The EC suspension thus obtained was . washed several times with RPM I 1640 (GIBCO) supplemented with 10% heat-inacti­vated fetal calf serum (FCS; GIBCO). The viabili ty ofEC as determined by trypan blue exclusion ranged between 80 and 90% in all experiments. In some experiments the EC suspension was incubated f?r 30 min at

Vol. 79, No.2

37°C in FCS-supplemented medium containing 12.5 J.lg/ml mitomycin C (Sigma Chemical Corp., St. Louis, Mo.) and, then, extensively washed before being used for cultures.

Lymph node lymphocytes: Popliteal, cubital, axillary, and cervical lymph nodes from Balb/c and C57Bl/6 mice were disrupted on a wil"e sieve and the cell suspension washed 3 times in RPMI 1.640 supple­mented with 10% heat-inactivated FCS. In order to obtain a cell population highly enriched for T lymphocytes, the washed cells were passed over nylon wool columns (FT 242 from Fenwal Labs. , Morton Grove, Ill.) in a slight modification of the procedure described by Schwartz, Jackson, and Paul [20]. Nonadherent cells were eluted, washed, and incubated for 30 min at 37°C to remove most of the residual adherent cells. The eluted cell population consisted of more than 95% T cells with contaminating cells consisting of B cells and 0.1-0.5% esterase-positive cells. This suspension was adjusted to a concen­tration of 2 X 10" viable cells/ml culture medium.

Culture Medium

In all experiments, RPMI 1640 supplemented with 20 mM L-gluta­mine (GIBCO), 10 mM Hepes buffer (GIBCO) , 2.5 X 10- 5 M 2-mercap­toethanol, 50 J.lg/ml gentamicin (Flow Labs., Bonn, FRG) and 2% heat­inactivated mouse serum was used. Sera from different mouse stra ins were tested in pilot experiments. Since serum from male NZB mice produced maximum proliferative responses, it was used throughout the study.

Antisera

(B10.A X A)F,-anti B10 serum (complement-dependent cytotoxic anti-Ia.8 titer = 1:32 on H-2") was a generous gift of Dr. D. Sachs (NCr, Immunology Branch, NIH, Bethesda, Md.). It was used in combination with LowTox rabbit-complement (C';Cederlane Labs. Ltd, Ontario, Canada) to eliminate la-positive cells from EC-suspensions. EC were incubated in RPMl 1640 containing the 1:10 diluted antiserum for 30 min at 4°C. C' was then added to a final dilution of 1:10 and the incubation continued for another 30 min at 37°C. Control cells were treated with equivalent dilutions of heat-inactivated normal mouse serum (NMS) and C'. Cells were then extensively washed and resus­pended to 2 X 10" viable cells per ml for further procedures.

A monoclonal IgM-anti-Thy 1,2-antibo<;ly (purchased from New Eng­land Nuclear, Boston, Mass.) and a FITC-goat antimouse-IgM antibody (Meloy Labs., Springfield, Va.) were used to determine the purity of the responder T cell population using established immunofluorescent techniques.

UV-B-Irradiation

6 X lOll EC/3 ml PBS were placed in 60-mm polystyrene Petri dishes (Nunc, Kamotrys, DK.). Sylvania F20 T12 fluorescent light bulbs emitting a continuous spectrum from 290-320 nm (= UV-B) served as irradiation source. Energy doses from 1 to 40 mJ / cm2 were administered in single doses as meascred at the surface of the cell suspension by the UV-B monitor (max. sensitivity at 297 nm) of an IL 700 Research Radiometer (Newburyport, Mass) . After irradiation, the EC were washed, their viability determined by trypan blue exclusion, and 2 X 10" viable cells resuspended per ml culture medium. Control cells were handled in the same way as UV-B exposed EC but received no UV.

Epidermal cell- lymphoclte reaction (ELR)

Triplicate cultuJ-es of 2 X 10" UV-B irradiated or non irrad iated, mitomycin C-treated or untreated, anti-Ia.8 exposed or unexposed EC, and equal amounts of syngeneic or allogeneic responder T cells were established in round-bottom wells containing 0.2 ml culture medium/ well. Wells containing pUl"ified T cells alone were used to evaluate background proliferation. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 1 to 14 days. Twelve hours before the termination of the culture period, 1 !LCi of tritiated thymidine (lH - TdR; 'specific activity 6.7 Ci/mM; New England Nu­clear) was added to each well. Cells were harvested with the aid of a multiple automated sample harvester (MASH II, Microbiological Ass. , Walkersville, Md.) and the radioactivity incorporated in to cellular DNA was determined by liquid scintillation counting. The results of triplicate cultures were expressed as counts per min (cpm)± standard error of the mean (SEM).

Assessment of Cell Viability after UV-Irradiation

Trypan blue exclusion: Irradiated or nonirradia ted EC were incu­bated in 17 X 100 mm test tubes (Falcon, Oxnard, Ca.) at 1 X lOn cells/ ml cuiture medium. At various time periods, aliquots of these cell

Aug. 1982 UV EFFECT ON LANGERHANS CELL-INDUCED T CELL ACTIVATION 131

suspensions were removed and cell viability determined by means of trypan blue exclusion.

Protein synthesis capacity: 10" irradiated or nonirradiated EC/ 0.2 ml culture medium were incubated on Nunc Microtest plates under ELR-culture conditions for various periods of time ranging from 0 to 48 hr. 12 hr prior to the termination of the culture 5 J.tCi of "H·leucine (L­: •. 4. 5-"H(N)-leucine, 120 CijmM; New England Nuclear) was added to each well. "H-leucine incorporation was determined by liquid scintilla­tion counting and used as a correlate for the protein synthesis capacity ofEC.

RESULTS

Murine Epidermal Cells Stimulate Activation of Syngeneic and Allogeneic T Lymphocytes

When highly purified T lymphocytes from either C57Bl/6 or Balb/c animals were cultured without additional cells from 1 to 14 days, only minimal DNA-synthesis occurred. A substantial proliferation was observed when Balb/c T cells were cocultured with either untreated (Table I) or mitomycin C-treated Balb/c EC but the amount of proliferation was approximately 25-40% less when mitomycin C-treated cells were used (data not shown). The proliferative T cell response in this syngeneic ELR (sELR) peaked on days 7-9 (Fig 1) .

A several-fold higher response than that observed under syngeneic conditions was seen when Balb/c EC were cocultured with allogeneic C57Bl/6 T lymphocytes (Table I). Vigorous proliferation was seen both with untreated or mitomycin C­treated EC and again, the use of untl'eated EC yielded a higher response. The time kinetics (Fig 1) of this allogeneic ELR (aELR) peaked on days 5 or 6.

Langerhans Cells, but No other Epidermal Cells, Are the Critical Stimulator Cells in sELR and aELR

The viability of freshly prepared EC usually ranged between 80-90%. After addition of either NMS plus C', or even C' alone, cell viability regularly dropped to a level of approximately 45-55%. Although formal proof is lacking, we believe that this phenomenon is attributable to the nonspecific killing potential of C' for some EC. No greater reduction in cell viability was seen when specific anti-Ia sera plus C' were added to EC; in other words, anti-Ia sera plus C' did not exert a discernibly greater killing effect than that seen with NMS plus C' or C' alone. On the other hand, pretreatment of EC and anti-Ia sera plus C' as opposed to pretreatment with NMS plus C' essen­tially abrogated their capacity to stimulate syngeneic or allo­geneic T cell proliferation as assessed on days 8 and 5, respec­tively (Table II) . We regulru'ly observed that the degree of T cell proliferation after pretreatment of stimulator cells with NMS plus C' was slightly less than that seen when untreated stimulator cells were used (data not shown).

We conclude from these experiments that fl;lllctionally rele­vant quantities of Ia antigens are expressed only by a small EC

TABLE 1. Mlirine EC stimulate activation of syngeneic and allogeneic T lymphocytes"

Exp. SLimulaLoI' cell Responder T cell cpm ± SEM"

1 None Balb/c 308 ± 42 Balb/c EC Balb/c 5.0]6 ± 243 Balb/c EC C57BI/6 31.753 ± 3.174

2 None Balb/c 431 ± 129 Balb/c EC Balb/c 6.232 ± 1.08i< Balb/ c EC C57BI/6 34.540 ± 3.704

3 None Balb/c 1.125 ± 640 Balb/ c EC Balb/c 13.439± 817 Balb/c EC C57BI/6 92.698± 6.036

"Culture conditions as described in "Materials and Methods," i.e., responder T lymphocytes were cultlU'ed alone 01' co-cultured with either syngeneic 01' allogeneic nonil'l'adiated EC. b Mean :'H - TdR - cpm ± SEM for triplicate cultlU'es.

cpm 10-5

o L-__ -_*~::-_._--.._.:....._=.. ~===*'=. _=_:::.~-=!!..-=-..!===!....-=_=*=*:!L.......J 6 7 8 9 10 11 12 13 14

DAYS IN CU LTURE

FIG 1. Kinetics of the syngeneic (opened circles ) and allogeneic (closed circles) ELR. Representative experiment. 2 x 105 nylon wool purified mUl'ine T cells were cultlU'ed alone (stars) or with equal numbers of syngeneic or allogeneic EC for various lengths of time. T cell proliferation was measured by 3H-TdR uptake and data are ex­pressed as the mean cpm of triplicate cultures ± SEM.

subpopulation (most likely LC [21]) and that this la-positive EC subpopulation is critical for the induction of syngeneic and allogeneic T cell proliferation.

UV-B Irradiation of EC Leads to a Dose-dependen.t Decrease in Syngeneic and Allogeneic T Cell Proliferation.

Irradiation of EC suspensions with varying doses of UV-B had profound effects on their capacity to initiate syngeneic and allogeneic T cell proliferation. Table III illustrates a represent-9.tive experiment in which Balb/c EC were either nonin:adiated or irradiated with UV-B doses ranging from 1 to 40 mJ/cm2

and then used as stimulator cells in both sELR and aELR. The T cell proliferative response was assessed on days 8 and 5, i.e., at times of maximal DNA synthesis in sELR and aELR, re­spectively. Data obtained in this experiment showed that 10 mJ/cm2 UV-B sufficed to essentially abolish both allogeneic and syngeneic T cell proliferation.

Pooled data from 4 experiments showed that a small dose of 1 mJ/cm 2 UV-B reduced the syngeneic T cell response by 10% ± 2.2 and the allogeneic by 23.7% ± 11.3 (Table IV). After 2.5 mJ/cm2 UV-B, the syngeneic response was lowered by 36.5% ± 11.5 and the allogeneic by 32% ± 2. After exposme to 5 mJ/ cm2 UV-B, both syngeneic and allogeneic ELR were reduced by about 62% and 10 mJ/cm2 neru·Jy abolished both the,synge­neic (83.8% ± 4.9) and the allogeneic (96.2% ± 1) ELR. Virtually no T cell proliferation above background levels were seen after doses of either 20 mJ/cm 2 or 40 mJ / cm2

.

Effect of UV-B Irradiation on EC-Viability

In order to determine whether the effect of UV -B irradiation on the ELR was due to a non-discriminatory effect of UV on all

132 ABERER ET AL Vol. 79, No_ 2

TABLE II. Effect of anti-fa serum plus C' on the stimulatolY capacity of EC in T cell prolifemtion"

Stimulator cells

None Balb/c EC Balb/c EC

None Balb/c EC Balb/c EC

Pre t.reatme nt

None NMS+C anti-Ia.8+C'

None NMS + C' anti-Ia.8+C'

Responder T cells

Balb/ c Balb/c Balb/c

C57BI/G C57BI/G C57BI/6

cpm + SEM" /), cpm % reduct.ion'

234 ± 71 3.GG7 ± 821 3.433

278 ± 49 44 99

25G ± G4 18.G41 ± 1.195 18.385

701 ± 47G 445 98

" Culture conditions as described in "Materials and Methods," i.e. round bottom culture plates with equal amounts of nonirradiated stimulator­EC and syngeneic or allogeneic T cells, harvested on day 8 and 5, respectively.

/, Mean "H-TdR-cpm ± SEM for triplicate cultures . .. Percent reduction was computed according to the formula:

[. cpm anti-Ia.8+C' -exposed EC ] 1 - . C' x 100. cpm non-antl-la.8+ exposed EC

TABLE Ill. UV-B induced, dose dependent abrogation of the capacity of EC to stimulate syngeneic and allogeneic T cell

proliferation: R epresentative experiment"

Stimulator ce lls"

Balb/c EC

Balb/ c EC

UV-B dose Responder T (mJ /cm' ) cells"

0.0 1.0 2.5 5.0

10.0 20.0 40.0 0.0 1.0 2.5 5.0

10.0 20.0 40.0

Balb/c

C57BI/6

cpm ± SEM"

G.032 ± 1.088 5.409 ± 1.054 4.519 ± 1.516

603 ± 156 4G2 ± 165

95 ± 38 77 ± 22

34.34Q ± 3.704 26.154 ± 3.105 22.554 ± 2.323 11 .773 ± 2.636

l.213 ± 299 269 ± 144

35 ± 49

% re­ducl ionf

>

11 25 90 92 98 99

24 35 66 96 99

100

" Culture conditions as described in Materia ls and Methods, culture period 8 days (sELR) and 5 days (aELR).

" Nonirradiated or UV -B-irradiated in doses ranging from 1.0 to 40.0 mJ /cm2 EC.

,. Column-passed syngeneic or allogeneic lymph node cells. " Mean "H-TdR cpm ± SEM for triplicate cultures. ,. Percent reduction was computed according to the formula:

1 - X 100. [ cpm UV-B-exposed EC ]

cpm non-UV-B-exposed EC

\ EC or due to a ?referential alteration of LC I-region function, we checked EC viability at different time periods after irradia­tion by trypan blue exclusion and, in addition, we tested their protein synthesizing capacity with a 3H-Ieucine incorporation assay.

80-90% of freshly prepared EC either nonirradiated or irra­diated with up to 20 mJ/cm2 UV-B excluded trypan blue indicating that the UV-B in the dose range applied did not produce immediate lethal cell damage_ In agreement with re­ports from the literature [22] that an epidermal cell suspension culture is a "dying system" we found that after 24 hr in culture, the viability of nonirradiated EC as well as those which had been exposed to up to 20 mJ/cm2 UV-B was nearly identical, but in each case had decreased to about 50%. After a 2-day culture period, both nonirradiated and UV -B-exposed cells were from 38-45% viable_ The viability of both UV-exposed EC a nd controls continued to decrease slowly on days 3 and 4 and on the 5th day of culture approximately 15% of the cells in each sample excluded trypan blue. Thus, whereas cell viability as assessed by trypan blue exclusion gradually decreases with increasing culture time, this reduction of viability is not accel-erated by UV irradiation. .

By contrast, in the 3H-leucine assay system, differences be­tween UV-exposed and nonexposed cells did emerge. Non-UV­exposed EC displayed a time-dependent reduction of protein­synthesizing capacity after various time periods in culture. After

TABLE IV. Dose dep endent abrogation of the capacity of EC to stimulate allogeneic and syngeneic T cell proliferation:" M ean value

± SEM of the percent reduction of four experiments

Stimulator cells"

Balb/ c EC

Balb/ c EC

UV-B-dose mJ / cm"

1.0 2.5 5.0

10.0 20.0 40.0

1.0 2.5 5.0

10.0 20.0 40.0

Responder T mean va lue ± SEM cells"

Balb/c

C57B1/ 6

10.0 ± 2.2 36.5 ± U .5 61.6 ± 19.7 82 .8 ± 4.9 94.0 ± 3.0 98.3 ± 0.3 23.7 ± 11.3 32.0 ± 2.0 61.8 ± 5.3 96.2 ± 1.0 98.4 ± 0.4 99.6 ± 0.4

" Culture conditions as described in Materials and Methods, culture period 8 days (sELR) and 5 days (aELR).

"Nonirradiated or UV -B-irradiated in doses ranging from 1 to 40 mJ/cm2 EC.

" Column passed syngeneic or allogeneic lymph node cells.

o 12 24 36 HOUR S

FIG 2. Effect of UV-B on the protein synthesizing capacities of murine EC. Pooled data from 6 experiments. From 1 to 40 mJ/cm2 UV­B-exposed or non-irradiated EC were cul tured at a density of 10" per 0.2 ml. 5 /lCi leucine were added at 0, 12, 24 , or 36 hI' of culture and after an additional 12 hr of culture "H-leucine incorporation was deter­mined by liquid scintillation counting. Bar graphs represent the "H­leucine uptake by UV-B-exposed EC as compared to nonirradiated control cultures (dotted line = 100%) after the equivalent culture time_

12 hr, protein synthesis was reduced by about 15% and by 60% after 24 hr; after 2 days of culture the amount of 3H-Ieucine uptake had dropped to between 10 and 20% of the original value_ A similar dose dependent reduction of protein synthesis was also observed after UV -irradiation but in contrast to the trypan blue assay, this reduction exceeded that observed in nonirradiated EC (Fig 2)_ Doses of 1.0 and 2.5 mJ/cm2 did not significantly affect protein synthesis when compared to the

Aug. 1982 UV EFFECT ON LANGERHANS CELL-INDUCED T CELL ACTIVATION 133

nonirradiated controls. 5 mJ/cm2 UV-B, which was shown to decrease the magnitude of the ELR by approximately 62% led to a decrease of 3H-Ieucine incorporation of only 22% after 12 hr, of 20% after 24 hr and 15% after 36 hr. 10 mJ/ cm2 UV-B which was shown to essentially abrogate both sELR and aELR led to a reduction of about 40% after 12, 24, and 36 hr. After 2 days, the amount of 3H-leucine incorporation by both non-UV­exposed and UV -exposed EC had dropped to a level at which meaningful comparison was no longer possible.

Thus, while a UV-B dose of, for instance, 10 mJ/cm2 essen­tially abrogates sELR and aELR, as shown above, it does not adversely influence EC viability over the course of the culture and only partially affects EC protein synthesizing capacities. These observations hold, in a dose-dependent fashion, for the entire UV -B dose-range employed.

DISCUSSION

Considerable controversy exists in the literature with regard to the nature of the stimulator cell type in both the syngeneic and allogeneic murine primary MLR. Both T [6,23] and B lymphocytes [23,24] were originally thought to be potent stim­ulators of the aMLR, whereas in the case of the sMLR, B lymphocytes have been implicated as the predominant stimu­lator cells [23,25]. These concepts have been strongly chal­lenged in the past few years. Ahmann et al [26] provided evidence that non-T radiation-resistant, poorly phagocytic, Ia­positive splenic adherent cells, but not T or B lymphocytes were mainly responsible for allogeneic T cell stimulation. Other investigators concluded from their experiments that certain murine dendritic cells, "Steinman cells" [7] which are known to reside in the spleen but are possibly also present in other lymphoreticular tissues represent virtually the only cell type capable of stimulating vigorous syngeneic and allogeneic MLR [27,28].

In the present report, we have demonstrated that there exists another cell type in the mouse which is a potent stimulator of sMLR and aMLR, i.e., the epidermal Langerhans cell (LC) . Although dendritic cells as described by Steinman and Nussen­zweig [7] share many features ofLC, certain differences between these 2 cell types do exist. Both are highly dendritic in shape and poorly phagocytic, both are derived from the bone marrow and both express I-A and I-E/C encoded la antigens [7,29-31] but lack surface Ig and Thy-l antigens. However, in contrast to Steinman cells, LC express easily detectable Fc-IgG receptors [32] and do not adhere to glass sill·faces. Moreover, the 2 cell types have different histochemical features and Steinman cells lack LC granules [33,34]. Although it is conceivable that LC and Steinman cells belong to the same lineage, such an onto­genetical relationship has never been experimentally estab­lished.

Our contention, that, among EC, LC are the critical accessory cells in the generation of syngeneic and allogeneic T cell reac­tivity, is based upon experiments in which EC were pretreated with either NMS plus C' or specific anti-Ia sera plus C'. In the former situation, substantial T cell proLiferation occurred, al­though to a somewhat lesser extent than that observed when untreated EC were used. The reason for the slight decrease in T cell proliferation after treatment of EC with NMS plus C' is most likely due to the fact that this pretreatment, or, even pretreatment with C' alone, results in a considerable reduction of EC viability. In order to include the same number of viable EC in all samples assayed, larger cell numbers had to be present in wells containing C'-treated EC than in those containing untreated EC, and we therefore assume that the decreased proliferation seen after C'-treatment may be attributable to steric factors or alterations in the culture milieu. On the other hand, pretreatment of EC with specific anti-Ia sera plus C' produced a decrease in EC viability which was not measurably different from that observed after pretreatment of EC with NMS plus C' or C' alone. Nevertheless, T cell proliferation was essentially abolished by anti-Ia plus C' in both the allogeneic

and syngeneic situation. This fmding implies that a small Ia­positive EC population is solely responsible for T cell prolifer­ation and, based on previous fmdings [21], we believe that it is the LC.

The mechanism by which LC act as potent stimulators of the ELR can only be considered in general terms. In the allogeneic situation, evidence exists that the I region of the MHC encodes the antigenic determinants (Ia antigens) responsible for stimu­lating most of the proliferative activity in the MLR [35]. It is therefore quite likely that LC Ia antigens are mainly responsible for allogeneic T cell activation, but perhaps, not exclusively so. Since the experiments presented in this paper were conducted in mouse strains disparate at the entire H -2 complex, the contribution of K and D region products to ELR stimulation cannot be ruled out [8]. The observation that pretreatment of EC with anti-Ia sera plus C' completely abrogated their ELR stimulatory capacity leads us to the assumption that la-positive accessory cells are needed for an ELR towards K, D, and I region differences to occur. In other words, the stimulator function of LC in a primary allogeneic ELR may not only be dependent on their expression of Ia antigens but also on their capacity to present alloantigens to the responding T lympho­cyte. One can further assume that MIs (minor lymphocyte stimulation locus) determinants [36] do not contribute to the allogeneic T cell proliferative response since mouse strains employed-while H-2 disparate-bore identical Mis alleles. We should also point out that the presence of MIs determinants on EC has never been formally proven.

While the mechanism of the sMLR is still poorly understood, it is generally agreed that la expression by the stimulator cell is an important prerequisite for this reaction [8]. Currently, 2 lines of thought are favored. One concept postulates that T lymphocytes respond directly to the I-region product indepen­dently of other antigen whereas another theory proposes that the sMLR is mediated by antigen present in the animal before sacrifice and carried over into tissue culture. Although our experiments do not prove or exclude either of these theories, it was of interest that the amount of T cell proliferation observed was highly dependent on the serum source used. Whereas only poorT cell stimulation was observed when isologous serum was used, the highest responses were obtained when sera from NZB mice were employed (see Materials and Methods). We can therefore not exclude the possibility that T cell stimulation by syngeneic LC is due to the presentation of newly introduced antigenic determinants into the culture system.

A peculiar feature of the LC-induced ELR is that its kinetics are different from that reported for experiments in which pe­ripherallymphoid cells or dendritic spleen cells are used. While in a conventional aMLR maximum stimulation is seen on the fourth day of culture [27] the LC-induced aELR consistently peaked on the fifth or sixth day. Similarly, conventional primary sMLR in the murine system are reported to peak at day 4-5 [28], whereas we observed maximum blastogenesis occurring on the eighth or ninth day in the LC-induced sELR. We do not know the reasons for these differences in kinetics. Initially, we considered the possibility that trypsinization of EC might have reduced the amount of Ia antigens present on LC and that the time needed for resynthesis of sufficient quantities ofIa antigens would therefore account for the delayed peak responses in the LC-induced ELR. In the meantime, we have obtained evidence that trypsinization is most likely not responsible for a change in T cell stimulation kinetics in that an MLR, which we estab­lished between trypsinized spleen cells and T cells, peaked on the very same day as an MLR established between unaltered spleen cells and T lymphocytes (data not shown) .

The fmding that UV irradiation of epidermal cells led to a dose-dependent abrogation of their capacity to stimulate syn­geneic and allogeneic T lymphocytes was not entirely unex­pected, since previous reports had indicated that normal lym­phocytes do not proliferate in response to UV -treated lymphoid stimulating cells [10-12,37,38]. The mechanism by which UV-B alters ELR- and MLR-stimulator cell function still requires

134 ABERER ET AL

elucidation but 2 possible explanations for this phenomenon can be offered. Bach, Bach, and Sondel [13] proposed that the mouse MHC encodes for certain alloantigens (LD antigens) located mainly in the I region but also present in the K and D regions and that these LD determinants are primarily respon­sible for activating Ly 1 + proliferating helper T cells as is evidenced by vigorous thymidine incorporation in MLR assays. Following this line of thought one may argue that the reason why UV irradiation of stimulating cells abrogates their ability to evoke a proliferative ELR response is that it ablates or, at least, alters LD expression, in particular la antigen expression on LC. Although we have shown that UV-B irradiation in vivo leads to a dose-dependent disappearance of anti-la reactivity of human LC [39], studies in the mouse system seem to indicate that UV-B irradiation of EC in vitro is not paralleled by a preferential loss of LC la a ntigens [40]. Therefore, at least with EC, we do not have sufficient evidence to conclude that inter­ference of UV with LD determinants is solely responsible for UV-induced abrogation of ELR stimulator function.

There exists an alternative possibility to explain the interfer­ence of UV with ELR reactivity. As discussed above, the induction of a murine primary MLR can be viewed as alloan­tigen presentation by an la-positive accessory cell [9]. From studies in congenic mice, we know that a primary MLR can be readily elicited when stimulator and responder cells bear the same I-region specificity and differ only in their K and D region antigens [9]. If the inductive stimulus for T cell proliferation depended only on alloantigen expression on viable stimulator cells, one would expect that K and D region bearing keratino­cytes initiate substantial allogeneic T cell proliferation. Our experiments show that this is not the case in that the induction of a primary ELR by epidermal stimulator cells is critically dependent on the presence of la-positive LC. It, therefore, seems reasonable to assume that the abrogation of ELR stim­ulator function is due to a particular UV -sensitivity of LC alloantigen-presenting functions rather than due to a selective interference of UV with alloantigen presentation. This is in keeping with three other observations suggesting a particular UV-B sensitivity of antigen presenting functions: UV-irradia­tion in vivo is followed by a loss of splenic adherent cell antigen presenting function without a concomitant alteration of B or T cell function [16]; in vitro UV -B-irradiated EC lose their capac­ity to present soluble protein antigens to immune T cells [19]; and UV-treated, haptenized EC do not induce allergic contact sensitization when injected subcutaneously in syngeneic recip­ients whereas nonirradiated EC do [40].

The finding tl;lat UV-B irradiation of epidermal cells abro­gates their ELR stimulatory capacity may prove to be a useful strategy to modify immune responses initiating in the skin, particularly in view of the additional finding that UV -B doses which eliminate LC accessory cell function do not produce lethal damage to their epidermal symbionts.

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