Eur. J. Immunol. 1992. 22: 581-586

Aimin Tang and Mark C. Udey Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda

Cultured Langerhans cells retain function after exposure to UVB irradiation or PFA

581

Differential sensitivity of freshly isolated and cultured murine Langerhans cells to ultraviolet B radiation and chemical fixation Previous studies have demonstrated that low doses of ultraviolet B (UVB) radiation (100 J/m2) abrogate the accessory function of freshly isolated murine epidermal Langerhans cells (fLC) and cause a parallel decrease in the ability of LC to express increased amounts of ICAM-1 (CD54) in vitro. We have subsequently observed that the accessory cell function of cultured LC (cLC), as assessed by their ability to support anti-CD3 monoclonal antibody (mAb)induced T cell mitogenesis, was not inhibited by levels of UVB exposure (100 J/m2) that completely inhibited the function of fLC, although exposure of cLC to UVB radiation (100 J/m2) resulted in a decrease in the level of ICAM-1 expression on most cLC and a concomitant decrease in cLC survival during a subsequent 24-h incubation. Time course studies revealed that T cells stimulated with antLCD3 mAb in the presence of cLC became committed to proliferate 4-8 h after culture initiation, while 24-30 h of co-culture was required for irreversible T cell activation when fLC were utilized as accessory cells. In addition, paraformaldehyde (PFA)-fixed (non-viable) cLC supported antLCD3 mAb-induced T cell proliferation, whereas PFA-fixed fLC were ineffective. We propose that cLC are functionally resistant to low doses of UVB radiation and chemical fixation because cLC express sufficient levels of the adhesion or co-stimulatory molecules [including ICAM-1 and Mac-1 (CDllb/CD18)] required to induceT cell activation. Conversely, fLC are sensitive to the effects of UVB radiation and chemical fixation because these physicochemical agents prevent acquisition of critically important surface molecules in culture.

1 Introduction Local suppression of cutaneous immune responses by ultraviolet B radiation (UVB) may result from direct untoward effects of UVB on Langerhans cells (LC) and reflect a diminution in accessory cell activity following exposure of LC to UVB in vivo or in vitro [1-41. Functional impairment of LC has been attributed to inhibitory effects of UVB on several cellular metabolic capabilities, including class I1 MHC antigen expression [5], cytokine production [6] and protein antigen processing [7]. We have recently focused on the effects of UVB on LC adhesion molecule expression in vitro, and have suggested that many of the inhibitory effects of UVB on LC function can be explained by selective effects of low levels of UVB on ICAM-1 expression by freshly isolated LC (fLC) [4]. Adhesion molecules are known to be important determinants of accessory cell function. Cells normally devoid of accessory cell activity have been converted into potent protein antigen presenting cells by co-transfection with

[I 98641 Correspondence: Mark C. Udey, Dermatology Branch, National Cancer Institute, National Institutes of Health, Building 10,Room 12N254, Bethesda, MD 20892, USA Abbreviations: cLC: Cultured Langerhans cell EC: Epidermal cell fLC: Freshly isolated LC ICAM-1: Intercellular adhesion molecule-1 LFA-1: Lymphocyte function-associated antigen-1 PFA: Paraformaldehyde UVB: Ultraviolet B radiation 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

genes encoding class I1 MHC antigens and ICAM-1, for example [8]. Conversely, cells which ordinarily possess accessory cell activity are rendered functionally incompetent if surface levels of adhesion molecules are reduced as a result of mutations that occur in nature [9] or that are induced in the laboratory [lo]. I n selected instances, co-immobilization of adhesion molecules and T cell receptor ligands (such as anti-CD3 mAb) on solid surfaces is sufficient to cause T cell activation [11], suggesting that in addition to promoting physical interactions between T cells and accessory cells, adhesion molecules may transmit co-stimulatory signals from accessory cells to T cells. Although the roles that selected adhesion molecules play in immune responses in the skin and elsewhere are relatively well characterized, novel intercellular adhesion molecules continue to be identified [12] and interactions between previously described adhesion molecules are becoming better characterized [13]. The epidermal LC is uniquely suited for studies which relate accessory cell activity to expression or lack of expression of individual cell surface proteins (or other cellular properties) because these cells spontaneously undergo functional and surface phenotypic changes in vitro which perhaps can be related to their specialized role in vivo [14-161. Cultured LC (cLC; which may represent the in vitro counterparts of antigen-bearing LC that have migrated to regional lymph nodes) are substantially more potent accessory cells than fLC [14], although cLC from certain inbred strains of mice are relatively inefficient processors of protein antigens [17, 181. A change in the level or array of adhesion or co-stimulatory molecules expressed on the surfaces of cLC (as compared with fLC) 0014-2980/92/0202-0581$3.50+ .25/0

582

A. Tang and M. C. Udey

may explain, in part, why cLC possess enhanced accessory cell activity. Indeed, increases in the level of expression of ICAM-1 on murine cLC [4] and LFA-3 [19] and B7/BB1 [20] on human cLC as compared with fLC have been reported. Previous studies suggested that inhibition of LC accessory cell function by UVB might be directly attributable to inhibitory effects of UVB on ICAM-1 expression by fLC [4]. Based on these results, we predicted that cLC (which already expressed high surface levels of ICAM-1) would be insensitive to UVB. Studies described here demonstrate that cLC are indeed functionally resistant to levels of UVB that completely inhibit fLC accessory cell activity and also retain accessory cell activity after chemical fixation, although both exposure to UVB and fixation are cytotoxic. We suggest that the accessory cell activities of fLC and cLC are differentially sensitive to UVB and chemical fixation because cLC express sufficient quantities of the adhesion or co-stimulatory molecules required to facilitate T cell activation, whereas fLC must acquire these molecules in culture before they become functionally competent.

2 Materials and methods 2.1 Mice Female BALB/c (H-2d) mice were obtained from Harlan Sprague Dawley (Frederick, MD) and were used as a source of epidermal cells and spleen cells at 10-14 weeks of age.

2.2 Antibodies The hamster mAb 145-2C11 (anti-CD3 epsilon chain) [21] was provided by J. A. Bluestone (University of Chicago, Chicago, IL). The mAb M5/114.15.2 [anti-I-A(b, d, and q haplotypes) and anti-I-Edk, rat IgG2b], MAR 18.5 (mouse IgM anti-rat Ig, kappa chain specific), M17/4.2 (anti-LFA-1 alpha subunit, rat IgG2,), MU70.15.11.5.HL (anti-Mac-1 alpha subunit, rat IgG2b), 53-6.72 (anti-Ly-2, rat IgG2,), MU9.3.4 HL.2 (anti-CD45, rat IgG2,), and M1/89.18.7.HK (anti-CD45, rat IgG2b) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The rat mAbYNh.7.4 [22] (anti-mouse ICAM-1, rat IgG2,) was a gift from F. Takei (British Columbia Research Center, Vancouver, B.C., Canada). MK-D6 (anti-mouse I-Ad, mouse IgG2,) and an irrelevant mouse IgGza control mAb were purchased from Becton Dickinson (Mountain View, CA). Purified anti-mouse CD2 (RM2-4, rat IgG2b) mAb was obtained from PharMingen (San Diego, CA) and FITC-conjugated rat anti-mouse IgG (kappa chain specific) mAb was purchased from Zymed (San Francisco, CA). FITC-conjugated affinity-purified goat anti-rat Ig, biotinconjugated affinity-purified F(ab’)z fragments of goat anti-rat Ig (human and mouse serum absorbed), and R-phycoerythrin-conjugated streptavidin were purchased from Tago, Inc. (Burlingame, CA). 2.3 Preparation of epidermal cells Epidermal cell (EC) suspensions were prepared from mouse ear skin by limited trypsinization as previously

Eur. J. Immunol. 1992. 22: 581-586

described [3] and used as a source of fresh LC directly. EC were cultured in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10 YO(v/v) FCS (Biofluids, Rockville, MD), 1% (v/v) antibiotic-antimycotic (1OOX) (Gibco BRL), 2 mM L-glutamine (Biofluids), 10 mM Hepes (Biofluids), 1 mM sodium pyruvate (Gibco BRL), 0.1 mM non-essential amino acids (Gibco BRL) and 5 x 10” M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO) at 37°C in an incubator containing 5 YOCO2 as indicated. Nonadherent cells were subsequently recovered, layered over a dense Ficoll-Hypaque gradient [4] and sedimented at 400 x g for 10 min at room temperature. Interface cells were pooled, washed and used as a source of cultured LC. Viabilities were determined by trypan blue exclusion and ranged from 80 ‘70to 94 YOfor fresh EC and 62 YOto 88 ‘70 for cultured EC.

2.4 UVB irradiation and paraformaldehyde (PFA) fixation of EC Fresh and cultured EC were exposed to low levels of UVB radiation from a UVB source consisting of a bank of four FS20 Sunlamp bulbs (Westinghouse Corp., Pittsburgh, PA) as previously described [4]. Cells were subsequently washed once with 5 % FCS-HBSS, enumerated and added to cultures. The viability of EC, as assessed by trypan blue exclusion, was not acutely altered by UVB irradiation. Freshly isolated and cultured EC were chemically fixed with PFA, 1% (w/v) in PB5, during a 15-min incubation at room temperature as indicated. After fixation, cells were washed three times in 5 YO (v/v) FCS/HBSS and counted prior to addition into culture. PFA-fixed cells were uniformly permeable to propidium iodide.

2.5 Flow cytometry (FCM) and immunofluorescence To enumerate LC, EC were incubated sequentially with MK-D6 and FITC-conjugated rat anti-mouse IgG and analyzed by FCM (FACScan flow cytometer, Becton Dickinson). Propidium iodide was added to each sample prior to analysis and dead cells were excluded. Expression of ICAM-1 by LC was assessed as previously described [4]. FCM data was analyzed using Becton Dickinson FACScan Research or Consort 30 software. 2.6 Anti-CD3 mAb-induced T cell proliferation

Accessory cell-depleted splenic T cells were prepared as described previously [4,23] and co-cultured (2 X 105/well) with UVB-irradiated or sham-irradiated fresh or cultured EC in flat-bottom 96-well microtiter plates (Costar, Cambridge, MA; 0.2 ml/well) in RPMI 1640 supplemented with 10 YO (v/v) FCS, 1YO (v/v) antibiotic-antimycotic ( 1 0 0 ~ ) 2 mM glutamine, 10 mM Hepes, 5 X M 2-mercaptoethanol and indomethacin (1 pg/ml, Sigma). Anti-CD3 mAb (145-2C11) was added as hybridoma supernatant (1 % v/v final concentration). Cultures were incubated at 37 “C in 5 % COzfor 42 h and [jH]dThd (1 pCi/well) was added for the final 6 h of the culture period. [jH] dThd (25 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Labeled cells were harvested using a semi-automated cell harvester (PHD Cell Harvester, Cambridge Technology

Eur. J. Immunol. 1992. 22: 581-586

Cultured Langerhans cells retain function after exposure to UVB irradiation or PFA

Inc., Cambridge, MA) and cell-associated radioactivity was measured by liquid scintillation counting. In some experiments, mAb reactive with adhesion molecules (or isotype-matched control antLCD45 mAb) were added into culture and their ability to inhibit anti-CD3 mAb-induced T cell proliferation was determined. mAb concentrations are presented as volume YOwhen hybridoma supernatants were added and as pg/ml when purified mAb were added. Purified mAb were diluted with complete media, sequentially dialyzed against HBSS and RPMI 1640 and filter-sterilized before addition into culture.

3 Results 3.1 Cultured murine LC are functionally resistant to low levels of UVB radiation Freshly isolated and cultured EC were prepared from the ear skin of BALB/c mice, exposed to low levels of UVB (0-200 J/m2) in vitro as indicated and assayed for their ability to support the proliferation of splenic T cells in response to anti-CD3 mAb as described in Sect. 2.6. Exposure of fLC to levels of UVB as low as 100 J/m2caused virtually complete inhibition of accessory cell activity ([4] h

E

BOOOO

2

60000

A

f

i

E 0

40000

0 0 S

1000

10000

100000

583

and Fig. 1A). In contrast, the accessory cell activity of LC that had been cultured in vitro for 72 h before exposure to UVB was largely unaffected by levels of UVB as high as 200 J/m2 (Fig. 1B).

3.2 Effects of UVB radiation on ICAM-1 expression by CLC We have suggested that UVB inhibits LC accessory cell function because UVB-irradiated fLC express lower levels of ICAM-1 after a 24-h culture period than unirradiated LC [4]. Since cLC express high levels of ICAM-1 on their cell surfaces [4], the apparent functional resistance of cLC to UVB depicted in Fig. 1B was expected. To confirm the existence of a relationship between surface levels of ICAM-1 and sensitivity to the effects of UVB, we studied the effect of low-dose UVB on the expression of ICAM-1 by cLC. Freshly isolated and 72-h-cultured BALB/c EC were exposed to UVB (100 J/m2), cultured for an additional 24 h and ICAM-1 expression was assessed by FCM. As we have previously reported [4], ICAM-1 expression was low on fLC, but increased rapidly and markedly in culture. Exposure of fLC to UVB (100 J/m2) before culture blunted the increase in ICAM-1 expression that otherwise occurred in vitro. Approximately two-thirds of the UVB-irradiated cLC exhibited ICAM-1-specificmean fluorescence that was approximately ninefold lower than that of sham-irradiated cLC, and was intermediate between the level seen on fLC and LC that had been cultured for 24 h. A smaller, but distinct, subpopulation of U VB-irradiated cLC actually expressed higher ICAM-1 levels than unirradiated cLC. Only the latter subpopulation of irradiated cLC excluded propidium iodide. Three additional experiments confirmed that, whereas the recovery of viable (propidium iodideexcluding) UVB-irradiated class I1 MHC antigen-bearing cells from 24 h cultures of fEC was 102 k 2.6 YOof control, the recovery of viable class I1 antigen-bearing cells from 24 h cultures of UVB-irradiated 72-h-cultured EC was only 39.2 k 4.1 YOof control.

Langerhans Cells /Well h

Y

5007

I

B

Langerhans Cells / Well Figure I . Inhibition of LC accessory cell activity by low-dose UVB radiation in vitro. Results are expressed as the mean cpm f SEM of ['HI dThd (3H-TdR) incorporated in triplicate cultures. T cells alone incorporated 401 f 166 cpm and T cells plus anti-CD3 ( n o accessory cells added) incorporated 489 f 27 cpm. Solid circles, unirradiated EC; open circles, EC exposed to UVB radiation, 50 J/m2; open squares, 100 J/m2; open triangles, 200 J/m2; crosses, unirradiated E C and T cells, n o anti-CD3 mAb added. (A) Freshly isolated EC added as a source of accessory cells; (B) cultured EC added.

3.3 cLC facilitate rapid activation of resting T cells cLC are functionally resistant to levels of UVB which cause significant amounts of delayed cytotoxicity. These results can potentially be reconciled in several ways. First,T cells may be activated in the presence of UVB-cLC before LC cytotoxicity becomes apparent. Second, non-viable UVBcLC may be functionally active. Third, all of the accessory cell activity of UVB-cLC may be contained within the minor subpopulation of cLC that remains viable in culture. To address the first possibility, we utilized mAb directed against adhesion molecules to interrupt T cell activation at various times after culture initiation and compared the rates at whichT cells became committed to proliferate when fLC and cLC was utilized as accessory cells.We have previously reported that LC-dependent anti-CD3-induced T cell proliferation can be largely inhibited by individual mAb directed against LFA-1, ICAM-1 or CD2 when fLC are employed as accessory cells, while isotype-matched antiCD45 mAb were not inhibitory [4]. These results are confirmed in Fig. 2A. When cLC were employed as

584

A. Tang and M. C. Udey

Eur. J. Immunol. 1992. 22: 581-586

accessory cells in parallel experiments, single mAb were much less effective inhibitors of T cell proliferation (compare Fig. 2A and B). Concentrations of anti-LFA-1 mAb that completely inhibited fLC function inhibited the activity of cLC by only -50 % . Amounts of anti-ICAM-1 and anti-CD2 mAb that largely inhibited the activity of fLC had little effect on cLC. Partial inhibition of cLC-dependent T cell proliferation was observed when fivefold higher concentrations of anti-ICAM-1 mAb were tested, however (data not shown). Results presented in Fig. 2B also suggest that the integrin Mac-1 may play a role in LC: Tcell interactions. Although anti-Mac-1 mAb were not inhibitory alone at the concentration shown in Fig. 2, anti-Mac-1 mAb augmented the partial inhibition of cLC-dependent T cell proliferation seen with anti-LFA-1 alone and also augmented the more complete inhibition seen with antiLFA-1 and anti-CD2 in combination. Anti-Mac-1 mAb alone also caused partial inhibition of fLC and cLC accessory cell activity at five-fold-higher concentrations

mAb Added antiLCD3

Al

anti-CDJ+LC +anti-LFA-1 +anti-CAM-1 +anti-CDZ +anti-LFA-I/CDP +anti-Mac-1 +anti-LFA-l /Mac-1 +anti-CDUMac-1 +anti-LFA-1KDUMac- 1 +anti-CD45(2a)

(data not shown). Anti-LFA-1+anti-CD2+anti-Mac-l inhibited both fLC- and cLC-dependent anti-CD3 mAbinduced T cell proliferation by >90 % .

To determine the rate at whichT cells became committed to proliferate in the presence of fLC and cLC, cultures were initiated withT cells, anti-CD3 mAb and fLC (or cLC), and a cocktail of anti-LFA-l+anti-CD2+anti-Mac-l mAb (or a cocktail of isotype-matched anti-CD45 mAb in similar concentrations) was added at the times indicated. As shown in Fig. 3, anti-LFA-l+anti-CD2+anti-Mac-l mAb inhibited the activity of fLC by >95 % when added at culture initiation (0 h). Addition of the cocktail as late as 8 h after the initiation of culture caused a similar level of inhibition. T cell activation became progressively resistant to inhibition by these mAb in the period 16-32 h after culture initiation. The time course of T cell activation observed when cLC were used as accessory cells was significantly different. Anti-LFA-1+anti-CD2+ anti-Mac-1 mAb were again markedly inhibitory when added at the beginning of the culture period (Fig. 3). Addition of mAb at 2 h caused the same level of inhibition as addition at 0 h, but T cell activation was virutally irreversible at 16 h and only marginal inhibition was seen when mAb were added 8 h after culture initiation. We believe that these results are indicative of the rate at whichT cells became committed to proliferate, and not the time at which T cell: LC cluster formation became irreversible, because anti-LFA-1+antiCD2+anti-Mac-l mAb disaggregated clustered cells (data not shown). cLC may, therefore, cause T cells to become irreversibly committed to proliferate in response to antiCD3 mAb before the delayed cytotoxicity of UVB radiation for cLC is evident.

+anti-CD45(2b) 0

20000

40000 60000

BOO00 100000 I20000

-1

3H-TdR Incorporated (cpm) mAb Added

fs

2s

n I

anti-CD3 anti-CDJ+LC +anti-LFA-l

300000

+anti-ICAM-1 +anti-CDZ

I

+anti-LFA-l/CDP +anti-Mac-l +anti-LFA-l/Mac-l +anti-CDUMac-1

I

+anti-LFA-l/CD2/Mac-l

lOWW

1OMx)

0

0

100000

200000

300000

400000

500000

3H-TdR Incorporated (cpm) Figure 2. Inhibition of LC-dependent, anti-CD3 mAb-induced mitogenesis by anti-adhesion molecule mAb. Anti-adhesion molecule mAb were added individually (or in various combinations) intoco-culturesofT cells, fLC (4.8 X lo3) orcLC (4.64 X lo3), and anti-CD3 mAb, and the ability of these mAb to inhibit Tcell proliferation was assessed. Hybridoma supernatants were added at 10 YO (v/v) alone or in combination as indicated. Anti-CD2 mAb was added at 4 pglml final concentration as indicated. Combinations of isotype-matched irrelevant mAb did not inhibit Tcell proliferation (data not shown). Results are presented as the mean cpm k SEM of triplicate cultures. (A) fLC were used as accessory cells. (B) 72-h cLC were used as accessory cells.

0 0

10

20

30

40

50

Time of mAb Addition (h)

Figure 3. Time course of anti-CD3 mAb-induced T cell activation with fLC and cLC as accessory cel1s.T cells were co-cultured with 7.2 x lo3 fLC or 5.74 x lo3cLC in the presence of anti-CD3 mAb, and a cocktail comprised of anti-LFA-1 (10Y0 vh), anti-CD2 (4 pg/ml) and anti-Mac-1 (10 YOv/v) mAb was added at various time points after culture initiation (0 h). t3H]dThd was added at 44 h and cell-associated radioactivity was measured at 48 h. A mixture of isotype-matched anti-CD45 mAb in identical proportions was used as control. Results are expressed as the mean cpm k SEM of triplicate cultures. Solid lines, anti-adhesion molecule mAb added; dashed lines, control mAb added. Open circles, fLC used as accessory cells; solid circles, cLC added.

Eur. J. Immunol. 1992. 22: 581-586

Cultured Langerhans cells retain function after exposure to UVB irradiation or PFA

3.4 Non-viable cLC support anti-CD3 mAb-induced T cell mitogenesis To determine if non-viable cLC might remain functionally active, we exposed freshly isolated EC and EC that had been cultured for 72 h t o 1% (w/v) PFA and then assayed PFA-fixed cells for accessory cell activity. Cells that had been exposed to PFA uniformly failed t o exclude propidium iodide (data not shown). No accessory cell activity could be detected in preparations of fresh EC that had been exposed to PFA (Fig. 4). I n contrast, PFA-fixed cultured E C retained significant amounts of accessory cell activity, although PFA-fixed cLC were less potent accessory cells than unfixed cLC (data not shown). However, both PFAfixed fLC and PFA-fixed cLC continued to express epitopes reactive with anti-Ia and anti-ICAM-1 mAb. T h e fluorescence intensity of anti-Ia (or anti-ICAM-1) stained PFAfixed fLC o r PFA-fixed cLC was 275 % of that of similarly stained unfixed cells (data not shown).

4 Discussion We have previously reported that inhibitory effects of low-dose U V B on EC-dependent T cell proliferation result from direct effects of U V B on L C and have suggested that this may be attributable to effects of U V B on ICAM-1 expression by fLC in vitvo [4]. If the level of ICAM-1 expressed by L C was the primary determinant of accessory cell activity, we reasoned that cLC (which expressed high levels of ICAM-1 and perhaps other adhesion o r costimulatory molecules) would be functionally resistant t o UVB. Results of the present study demonstrate that the accessory cell activity of cLC is indeed functionally resistant t o U V B and is also relatively resistant to chemical fixation, although both physicochemical agents are cytotoxic to cLC. Time-course studies reported herein also indicate that cLC trigger T cell proliferation in response to anti-CD3 mAb within several hours of culture initiation, whereas fLC activate T cells at a much slower rate (Fig. 3). We propose that the delay in T cell activation that is evident when fLC are employed as accessory cells results because LC must express critical levels of adhesion o r co-stimulatory molecules on their surfaces before they become fully competent. The adhesion o r co-stimulatory molecules expressed by murine fLC and cLC remain incompletely characterized. We have previously shown that surface ICAM-1 levels on murine LC increase dramatically within 24 h in vitro and continue t o increase over a several-day-culture period [4]. Since anti-CD2 m A b inhibit LC-dependent T cell mitogenesis, we infer that murine L C also express LFA-3. Human L C have been previously shown to express increased levels of LFA-3 after several days in culture ([19] and S. Aiba, S. Shaw and S. I. Katz, personal communication).The role of the murine equivalent of human B7/BB1 [24] in LCdependent T cell stimulation also remains undefined. Since the accessory cell activity of cLC is relatively resistant t o inhibition with anti-ICAM-1 (or anti-LFA-1) m A b individually (Fig. 3), it seems likely that adhesion o r costimulatory molecules in addition t o ICAM-1 are critically involved in cLC function. This is the first study that suggests that Mac-1 (CD1 1b/CDl8), a leukocyte integrin expressed at compa-

-k

s

100000 1

f

80000-

/ /

'El

al c

60000-

a

? 5

585

2oooo

I

100

1000

10000

PFA-fixed Langerhans Cells Well

Figure 4 . Non-viable (PFA-fixed) cLC retain accessory cell activity. PFA-fixed freshly isolated EC: were comprised of 1.9% LC whereas fixed, cultured EC contained 12.2 YO LC. T cells alone incorporated 179 f 38 cpm and T cells + anti-CD3 incorporated 831 k 178 cpm of [3H]dThd. Open circles, PFA-fixed fEC added;

solid circles. PFA-fixed cEC added. rable levels on both fLC and cL,C ([16] and data not shown) plays a role in the interaction betweenT cells and accessory cells, although Mac-1-dependent binding of human neutrophils t o endothelial cells has been demonstrated [25] .While the nature of the cell surface counter-receptor for Mac-1 involved in cell-cell interactions has been somewhat uncertain, at least one cell surface ligand has been conclusively identified [26]. Diamond and coworkers have shown that COS cells transfected with human Mac-1 adhere t o purified, immobilized human ICAM-1, and that COS cells transfected with ICAM-1 adhere t o purified, immobilized Mac-1 [26]. Mac-1 expressed on the surfaces of L C may facilitate LC: T cell interactions by binding t o ICAM-1 on T cells. Since certain Mac-1-dependent cell-cell interactions cannot be completely inhibited by anti-ICAM-1 mAb, it has been suggested that Mac-1 also binds t o another, as yet unidentified, cell surface protein 1261. A myriad of potential interactions between adhesion o r co-stimulatory molecules on LC and T cells is possible. Important information about the function of individual adhesion molecules (or specific binding pairs) has already come from studies of interactions between cells made t o artificially express single types of adhesion molecules [S] and from studies of the interactions of cells expressing well-characterized adhesion molecules withh purified counter-receptors [26]. To fully understand the role that adhesion molecules play in the initiation and propagation of immune responses in the skin and elsewhere, it will be necessary to continue t o study accessory cells and responding cells which each express arrays of adhesion and co-stimulatory molecules. The functional transformation that epidermal LC undergo in vitro is dramatic and provides an interesting and in some ways unique system in which to ask fundamental questions about the relationships between adhesion o r co-stimulatory molecule expression and accessory cell activity. Theauthors thank Dr. Fumio TakeiforsupplyingthemAb YNII.7.4 and Dr.7. Stephen I. Katz and Alexander E n k f o r reviewing the manuscript.

Received August 10, 1991; in final revised form November 7, 1991.

586

A. Tang and M. C. Udey

5 References 1 Noonan, F. I?, Kripke, M. L., Pedersen, G. M., and Greene, M. I., Immunology 1981. 43: 527. 2 Aberer, W., Stingl, G., Stingl-Gazze, L. A., and Wolff, K., J. Invest. Dermatol. 1982. 79: 129. 3 Stingl, G., Stingl-Gazze, L. A., Aberer, W., and Wolff, K., J. Immunol. 1981. 127: 1707. 4 Tang, A. and Udey, M. C., J. Immunol. 1991. 146: 3347. 5 Aberer, W., Schuler, G., Stingl, G., Honnigsmann, H., and Wolff, K., J. Invest. Dermatol. 1981. 76: 202. 6 Sauder, D. N., Noonan, F. F!, DeFabo, E. C., and Katz, S. I., J. Invest. Dermatol. 1983. 80: 485. 7 Stingl, L. A., Sauder, D., Iijima, M., Wolff, K., Pehamberger, H., and Stingl, G., J. Immunol. 1983. 130: 1586. 8 Altmann, D. M., Hogg, N., Trowsdale, J., and Wilkinson, D., Nature 1989. 338: 512. 9 Kresnky, A. M., Mentzer, S. J., Clayberger, C., Anderson, D. C., Schmalsteig, F. C., Burakoff, S. J., and Springer, T. A., J. Immunol. 1985. 135: 3102. 10 Dang, L. H., Michalek, M. T., Takei, F., Benaceraff, B., and Rock, K. L., J. Immunol. 1990. 143: 4082. 11 Seventer, G. A. v., Shimizu, Y., Horgan, K. J., and Shaw, S., J. Immunol. 1990. 144: 4579. 12 Metlay, J. F!,Witmer-Pack, M. D., Agger, R., Crowley, M. T., Lawless, D., and Steinman, R. M., J. Exp. Med. 1990. 171: 1753. 13 Diamond, M. S., Staunton, D. E., Marlin, S. D., and Springer, T. A., Cell 1991. 65: 961. 14 Schuler, G . and Steinman, R. M., J. Exp. Med. 1985. 161: 526.

Eur. J. Immunol. 1992. 22: 581-586

15 Shimada, S., Caughman, S. W., Sharrow, S. O., Stephany, D., and Katz, S. I., J. Immunol. 1987. 139: 2551. 16 Witmer-Pack, M. D., Valinsky, J., Oliver, W., and Steinman, R. M., J. Invest. Dermatol. 1988. 90: 387. 17 Romani, N., Koide, S., Corwley, M., Witmer-Pack, M., Livingstone, A. M., Fathman, C. G., Inaba, K., and Steinman, R. M., J. Exp. Med. 1989. 169: 1169. 18 Aiba, S. and Katz, S. I., J. Immunol. 1991. 146: 2479. 19 Teunissen, M. B. M.,Wormeester, J., Krieg, S. R., Peter, I? J., Vogels, I. M. C., Kapsenberg, M. L., and Bos, J. D., J. Invest. Dermatol. 1990. 94: 166. 20 Mirando, W. S., Tubesing, K., and Elmets, C. A., Clin. Res. 1991. 39: 533. 21 Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 1987. 84: 1374. 22 Prieto, J., Takei, F., Gendelman, R., Christenson, B., Biberfeld, €?,and Patarroyo, M., Eur. J. Immunol. 1989. 19: 1551. 23 Romani, N., Inaba, K., Pure, E., Crowley, M., WitmerPack, M., and Steinman, R. M., J. Exp. Med. 1989. 169: 1153. 24 Freeman, G. J., Gray, G . S., Gimmi, C. D., Lombard, D. B., Zhou, L.-J., White, M., Fingeroth, J. D., Gribben, J. G., and Nadler, L. M., J. Exp. Med. 1991. 174: 625 25 Zimmerman, G. A. and McIntyre, T. M., J. Clin. Invest. 1988. 81: 531. 26 Diamond, M. S., Staunton, D. E., Fougerolles, A. R. d., Stacker, S. A., Garcia-Aguilar, J., Hibbs, M. L., and Springer, T. A., J. Cell Biol. 1990. I l l : 3129.

Differential sensitivity of freshly isolated and cultured murine Langerhans cells to ultraviolet B radiation and chemical fixation.

Previous studies have demonstrated that low doses of ultraviolet B (UVB) radiation (100 J/m2) abrogate the accessory function of freshly isolated muri...
636KB Sizes 0 Downloads 0 Views