lmmunologic study Experimental contact sensitivity and innate immunity Experimental contact seT1Sitivity: a model for both antigen (hapten)-speciflc and innate immune mechan-isms M. Kataranovski, L. Kandolf Sekulovic and D. Karadaglic ABSTRACT In the present study, experimental model of contact sensitivity to dinitrochlorobenzene (DNCB) in inbred AO rats was employed to determine both antigen (hapten)-specific as well as parameters of antigen- non-specific aspects of contact hypersensitivity (CHS) response. Following local epicutaneous applica- tion of 2% DNCB, increased spontaneous, hapten-stimulated and interleukin (IL)-2-driven draining lymph node cell (DLNC) proliferation was detected, reflecting the antigen (hapten)-specific aspect of CHS. Changes in the ratio of lymphocyte subsets were noted in DLNC in the sensitization phase of CHS. At the dose of DNCB employed for the elicitation of CHS in sensitized animals, increased activity of peripheral blood granulocytes (including activation, adhesion and cell survival) was detected. Collectively, these data demonstrate the activation of both antigen (hapten)-specific and innate immunity in contact hyper- sensitivity. The importance of studying both mechanisms in this same model has been discussed. K E y Introduction WORDS contact hyper- sensitivity, DNCB; lymphocytes, granulocytes, . rat Skin responds to toxic agents by various inflamma- tory/immunologic cascades of events leading to the in- duction of allergic or irritant contact dermatitis or hy- perkeratosis (1). A commonly used model in testing the · animal's or human's ability to mount a cutaneous im- mtme response is contact hypersensitivity (CHS) reac- tion to skin reactive chemicals, haptens. The afferent phase of the CI-IS response, the sensitization phase, is initiated by epicutaneous application of the hapten to the dorsal or abdominal skin, and it is characterized by the activation and division of hapten-specific T lympho- cytes in the regional lymph nodes and the appearance of effector cells in the lymph nodes and spleen (2). In the efferent phase, which follows a subsequent chal- lenge with hapten, e.g. epicutaneous application of the sensitizer to the skin of the ear, primed T lymphocytes are recrnited to the site of challenge where they pro- duce a variety of inflammatory mediators, amplifying a background inflammatory response into a more vi- gorous process. It is the classical manifestation of con- Acta Dermatoven APA Vol 10, 2001, No 3 ------------------------------- --- 8J Experimental contact sensitivity and innate immunity tact hypersensitivity that is measured as "ear swelling". Ear swelling is the early-recognizecl skin response to hapten application, characterized histologically by der- mal cell infiltration (3). Epicutaneous application of haptens is commonly used test-system for antigen-specific T cell-mediated immune responses. Another type of T cell-mediated cutaneous hypersensitivity is delayed type hypersensi- tivity (DTH), widely used in testing cellular immunity to antigens following their subcutaneous or intracler- mal injection. Both CHS and DTH responses are often referred to as equivalent reactions , with the terms be- ing used interchangeably and their distinctiveness re- flecting only the types of antigen used ancl their route of aclministration (2) . There is a growing bocly of evi- dence, however, that CI-IS and DTI-I are different reac- tions as there are data clemonstrating that clifferent cytokines (IL-10 vs. TNF-cx) regulate CI-IS ancl DTH, re- spectively ( 4), and that differences exist regarcling the complexity of effector cell participation pattern in these reactions (5). It was shown further that non-antigen specific, proinflammat01y effects ofhaptens contribute to the elicitation of CI-IS (6) highlighting the importance of mechanisms of innate immunity in this reaction. In this regarcl, the involvement of granulocytes in CI-IS was stressed as clepletion of these cells prior to elicitation inhibited the CI-IS expression (7). Moreover, it was lmmunologic study shown that sensitization with hapten resultecl in the enhancement of peripheral blood granulocyte activity (8). Taken together, these clata further support the hy- pothesis that contact sensitivity is not a classic clelayed type hypersensitivity ancl that it might be viewed as a cutaneous reaction in which both antigen (hapten)-spe- cific and non-specific, innate immune mechanisms are engaged. In the present study, the rat model of contact hyper- sensitivity to clinitrochlorobenzene (DNCB), developed previously in our laboratory (9), was employed to de- termine both aspects of the CHS response. As hapten sensitization occurs centrally, within draining lymph nodes (10) relevant parameters of draining lymph node celi (DLC) activity including spontaneous and hapten- stimulated proliferation and celi subset composition were determined following the sensitization. As an in- volvement of granulocytes in the elicitation was sug- gestecl (7) peripheral blood granulocyte activity follow- ing the elicitation of CHS was studied also. By using a rat model of CHS, limitations of the mouse CHS reac- tion regarding peripheral bloocl granulocyte accessibil- ity were circumvented. Thus, basic functional aspects of peripheral bloocl granulocytes (activation, adhesion ancl cell survival) cluring the elicitation phase of CI-IS were cletermined. Table l. Changes in the clraining lymph nocle cells during the induction of CHS reaction DLN cellularity (xlO" cells) TCR (alpha/beta)" CD4" CD8" CD4/CD8 Dendritic cells in DLC populationb Spontaneous proliferation rate of DLC (c.p .m. x 105) Hapten stimulatecl proliferation rate (Lic.p.m.)" Interleukin-2 stimulated proliferation rate (Lic.p.m.)" 13.32 1.88 84.42 ± 3.79 (57 ± 15.56) 55.12 ± 6.62 (58.64 ± 2.63) 21.8 ± 4.76 (111.25 ± 9.22) 1.97 ± 0.34 O.OS 0.03 437158 negligible 1357 29 70.88 13.88** 79.72 ± 2.88 (68.82 ± 1.82) 44.74 + 1.55*** (77.19 ± 3.24***) 31.22 ± 1.01** (130.24 ± 5.78**) 1.43 ± 0.02*** 0.36 0.06*** 1734724059*** 13158+752 205625780*** aValues are given as mean percentage of irnmunofluorescence positive cells SD and as mean SD of mean fluores- cence intensity (MFI) in parentheses; bValues are given asa mean percentage SD of dendritic cells per pool of clraining lyrnph nocles; cc.p.m. = total c.p.m. minus c.p.m. in meclium *p<0.05; **p<0.025; ***p<0.01 versus vehicle 86 -------------------------------------Acta Dermatoven APA Vol 10, 2001, No 3 Immunologic study Materials and methods The experiments were performed on inbred male Albino Oxford (AO) rats (Farm for Experimental Ani- mals, Milita1y Medica! Academy, Belgrade, Yugoslavia) in adherence to the NIH guidelines for the use of ex- perimental animals, with a permission of the Ethical Committee of our Institute. Groups of 6 to 8 rats re- ceivecl 100 µL of 2% w/v of 1-chloro-2,4-dinitrochloro- benzene, DNCB (BDH Chemicals LTD, Englancl) clis- solved in vehicle (acetone:olive oil 4:1) or an equal volume of vehicle solely, on the shaved dorsum for 2 consecutive days as described (9). Three clays follow- ing sensitization, the rats were challenged by an appli- cation of 50 µL of suboptimal close (0,66%) of DNCB to the miter half of the left ear. Ear thickness was mea- sured with an engineer micrometer 24 hours after the challenge. The intensity of the response was quantifiecl as the clifference in the thickness between the chal- lenged and non treatecl ears of the same animal ancl expressecl asa percent of increase in ear thickness. Catti- lage-free halves of exposecl ears were fixecl, stainecl with haematoxyllin and eosin for histological evaluation. Density of the clermal infiltrate was measured by the computer-assistecl image analysis system using software "MIKRO" (Laboratory for the computer systems, Insti- tute "Mihajlo PUPIN", Belgracle). Draining (axillary ancl suprascapular) lymph nocle celi activity ancl phenotype were cletermined 72 h fol- lowing sensitization. Suspensions of DLNC were pre- pared in the complete medium containing RPMI-1640 cul ture medi um (ICN Flow, ICN Biomedicals Inc., Costa Mesa, CA, USA) supplemented with 5% heat-inactivatecl fetal calf serum (FCS) (ICN Flow), 1 % gentamycin and Sx10·5 M 2-mercaptoethanol. DLNC were cultured for 24 hours in the meclium only or in the presence of 50 IU/ml of recombinant human IL-2 (Genzyme, MA, USA) (for spontaneous proliferation and IL-2-stimulated pro- liferation, respectively) or 4 clays in the presence of 5 µg/ml of clinitrobenzene sulfonic acid, DNBS (BDH Chem, UK), a water soluble analogue of DNCB (hap- ten-stimulatecl proliferation) at 37°C in a humiclifiecl at- mosphere containing 5% CO 7 • Proliferation was esti- matecl by incorporation of3H tl1ymidine (1 µCi/culture, Amersham, UK) aclclecl at the onset (spontaneous pro- liferation) or during the last 16-18 h of culture (IL-2- stimulated ancl DNBS-stimulatecl proliferation). Results are expressecl as counts per minute (c.p.m.) measurecl by P-raclioactivity scintillation counter (LKB RACK-Beta). For the flow cytometry suspensions of DLNC in phosphate bufferecl saline were incubatecl with mono- clonal antibodies R73 (anti-rat TCR cx/p), W3/25 (anti- rat CD4) , OX-8 (anti-rat CD8), all purchasecl from Serotec, Ltd, Bicester, UK, followed by rabbit anti Ig- FITC conjugatecl antibody (INEP, Zemun, Yu). Cells Experimental contact sensitivity and innate immunity were analyzed for fluorescence intensity on an FACScan flow cytometer (Becton Dickinson, Germany). Results are expressed as percentages of fluorescence positive cells. Peripheral blood granulocytes were isolatecl from the heparinized bloocl withclrawn from the abclominal aorta, 24 h following challenge, by clextrane seclimen- tation (6% w/v dextrane Pharmacia, Uppsala, Sweclen), clensity gradient centrifugation (NycoPrep Animal 1007, Nycomecl AS, Oslo, Norway) followecl by lysis of eryth- rocytes with isotonic NH 1 CI solution. The remaining granulocytes were washed twice, resuspenclecl in com- plete RPMI-1640 cul ture medi um ( without 2-mercapto- ethanol) ancl helcl at room temperature for functional studies. The purity of the neutrophils was more than 95%, as cleterminecl by May-Grunwalcl-Giemsa staining. A quantitative colometric assay clescribed for human granulocytes (11) in which tetrazolium salt MTT is meta- bolically reclucecl to colored end procluct, formazan, was employecl to estimate cell survival of rat peripheral bloocl granulocytes. MTT recluction was estimatecl im- mecliately (in freshly isolated cells) or following 18 h in culture. Formazan proclucecl was clissolvecl in SDS-C! ancl absorbency was measured at clual wavelength, 570nm/650nm by an ELISA 96-well plate reacler (Beh- ring ELISA Processor, Behring, FRG). Celi survival was expressed as MTT inclex (absorbance of formazan solu- bilizecl at the ene! of incubation period divided by ab- sorbance at the onset of culture). Spontaneous ancl phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) (Sigma Chemical Co., St. Louis, Mo., USA) stimulatecl activation of granulocytes was evalu- atecl by a cytochemical assay for the respirato1y burst (12) , which is the measure of an intracellular recluction of nitroblue tetrazolium (NBT). Formazan proclucecl by granulocyte recluction ofNBT was extractecl with acicli- fiecl SDS and absorbance measured spectrophotometri- cally as clescribecl above. Adhesion of granulocytes to plastic was assessed by using a moclifiecl assay initially described by Oez (13) in which the 60-minute adhe- sion of granulocytes to plastic of a 96-microwell plate was measured. After incubation in the medium (spon- taneous aclhesion) or in the presence of 50 ng/mL PMA and washing off 11011-adherent cells, adherent cells were stained with 0.1% methylene blue. The absorbance of acicl-clissolvecl clye was measured at 650 nm/570 nm by an ELISA 96-well plate reacler. Data are expressecl as mean value ±SD for each ex- perimental animal group (5-7 animals) for the cletermi- nation of ear swelling ancl DLNC ph,motype. In the DLNC ancl granulocyte functional assays, clata repre- sent means ±SE for triplicates or quadruplicates from single experiments representative out of three . Signifi- cance was clefinecl by Stuclent's t-test. p values less than O.OS were consiclered significant. Acta Dermatoven APA Vol 10, 2001, No 3 - ----- -------------------- ---- ----~ 87 Experimental contact sensitivity and innate immunity Results Ajferent phase ofthe contact hypersensitivity reaction: draining lymph node cell activity Changes in draining lymph node cells 72 hours fol- lowing local epicutaneous application of2% DNCB were determined as preliminary experiments had demon- strated a maximal spontaneous proliferation rate of DLNC at this tirne point. The data are summarized in Table l. A shift in the ratio of CD4+ to CD8+ DLNC was noted on day 3 following the DNCB application, due to a decrease in relative numbers of CD4+ T cells and an increase in relative numbers of CD8+. Mean fluorescence intensity of cell surface CD4 and CD8 antigens on DLNC was increased in the DNCB-treated group compared to the vehicle-treated group. As increasecl cellularity of draining lymph nodes of sensitizecl animals was noted, absolute numbers of cells from these subsets greately outnumberecl those in clraining lymph nodes of 11011- sensitized animals. An enrichment in cells with clistinct denclritic morphologywas observed in DLNC from sen- sitizecl animals. Significantly increased levels of spon- taneous proliferation as well as IL-2-stimulatecl and DNBS-stimulated proliferation were detected in cultures of DLNC from sensitized animals, which coulcl be as- cribecl to increasecl percentages of IL-2W cells in the sensitizecl compared to the vehicle treated animals (8.6±0.99 VS. 5.50±1.27). Ejferent phase qf contact hypersensitivity reaction Ear swelling Ear swelling assay as well as descriptive and quanti- tative histology were cletermined 24 hours following challenge with DNCB. Nearly a 10-folcl increase (108.2±39.3% vs. 9.51±5.96%, p<0.05) in ear swelling was notecl following the application of 0.66% DNCB. This increase was accompanied by an inflammatory re- sponse in the exposed skin characterizecl by epiclermal changes, similar to the well-known picture of allergic contact dermatitis, inclucling vacuolisation of the basal layer, and spongiosis with formation of microvesicles at the dermo-epidermal junction. Subtle superficial crusts were also seen. Density of the clennal infiltrate, measured by the computer-assistecl image analysis sys- tem, was increased compared to the control, vehicle treated group (22.81±0.81 cells/area vs. 13.67±3.4 cells/ area, p<0.05) in which ali of the describecl epiclermal ancl dermal changes were not seen. 88 lmmunologic study Peripheral blood granulocyte activity in the elicitation phase oj CHS Changes in peripheral blood (PB) granulocyte timc- tion were clemonstrated following a challenge with sub- optimal doses of DNCB and are presented in Table 2. Increased MTT inclex was notecl in cultures of granulo- cytes from sensitized animals. Both spontaneous ancl PMA-stimulated NBT recluction were increasecl follow- ing challenge with 0.66% DNCB. A stronger PMA-stimu- latecl aclhesion of granulocytes was notecl in DNCB- treatecl group. Discussion In this stucly, parameters of both hapten-specific as well as innate immune responses were cleterminecl in experimentally induced contact sensitivity in rats. En- richment in the clenclritic celi contents in lymph nodes that clrain the area of epicutaneous DNCB application is in concorclance with data from numerous studies conducted in mouse models of CHS which demon- stratecl the appearance of clenclritic cells in regional lymph nocle.c; following hapten application (2). An in- crease in cellularity of draining lymph nodes, a com- monly used parameter of local lymph node activity (14, 15) might be a result of either lymphocyte proliferation or local recruitment of antigen-specific lymphocytes or both. Lymphocyte proliferation is evident in vitro as increasecl spontaneous proliferation ancl ocurring in response to exogenous IL-2 ancl DNBS. Phenotypic characterization of DLNC showed in- creasecl numbers of clraining lymph node cells express- ing CD4 and CD8 antigens. This is in line with investi- gations linking the effector stages of CHS with CD8+ (16-19) and/or CD4+ T cells (5, 20). The upregulation of celi surface expression of CD4 may be in accordance Table 2. Changes in the granulocyte activity during elici- tation of CHS reaction MTT index 0,73±0,02 NBT spontaneous 0.054±0.0 PMA stimulatecl 0.199±0.001 Adhesion spontaneous 0.015±0.002 PMA stimulatecl 0.026±0.001 Values are given as mean ±SE *p<0.05 and ** p<0.01 versus vehicle 0,93±0,00** 040.066±0.001 * 0.235±0.002* 0.019±0.003 0.034±0.003* Acta Dermatoven APA Vol 10, 2001, No 3 lmmunolo g i c study with a study which found a correlation between CD4 density and the functional activity of CD4+ in the CHS response (5). Similarly, the observed increase in cell- surface density of CD8 might also be related to the in- volvement of the cells in the effector arm of CHS. On the basis of parameters of draining lymph node activity, as well as on the elicitation pluse of reaction, the rac model of CHS reaction presented is compatible with the mouse model and could be used, as recom- mended earlier, as a suitable model for cell-mediated immunity studies in experimental contact allergy (21). Recent studies have brought evidence on the in- volvement of innate immunity/ inflammatory responses in CHS. It was sh own that antigen non-sp ecific, proinflammat01y effects of hapten contribute to the elici- tation of CI-IS (6) and that the dual (irritant and anti- genic) properties of sensitizing chemicals are needed for the development of allergic contact dermatitis (22) . In this regard, the importance of granulocytes in the effector pluse of CHS was suggested (7). The rac model of CHS enabled analysis of periph- eral bloocl granulocyte function in the elicitation of CHS. The functional state of peripheral blood granulocytes in the elicitation phase of CHS was examined by in vitro tests of granulocyte activation, adhesion and survival. The potential of granulocytes to reduce tetrazolium salts to formazan via tetrazolium reducing respiratory burst oxiclase (23) was used as an in vitro measure of gram1- locyte activation (12). It showecl increased spontane- ous activation of granulocytes from challenged animals. Significantly higher responses provoked with subopti- mal PMA (50 ng/ml) in granulocytes from challenged animals compared to those seen in controls, suggest a primecl state of the granulocytes (24). Adhesiveness of granulocytes to various matrices is commonly used test of granulocyte activity, as an in vitro correlate of granulocyte adherence to endothe- lium or connective tissue matrices (13). Plastic microtitre plates are w idely used in the assessment of granulocyte adhesion as plastic surfaces bebave physiologically with respect to aclhesiveness of granulocytes inducecl by various stimuli (13, 25). There was no difference in spon- taneous adhesion of granulocytes from challengecl com- pared to vehicle-treated animals. However, significantly increased adhesion of granulocytes from challengecl vs control animals fo ll owing stimulation with PMA, a known stimulus of granulocyte adhesion (26), may re- flect a higher propensity of granulocytes from chal- lenged rats to adhere. The increase in activation of peripheral blood gram1- locytes following challenge with DNCB might have re- sulted from the cutaneous toxicity of DNCB and higher demands for granulocytes at the site of hapten applica- tion . Dermatotoxicity ofDNCB is well known (27, 28). Increased activity in recluction of NBT following elici- tation of CI-IS, might also resulted from previous activa- tion of peripheral blood granulocytes as demonstrated Experimental contact sensitivity and innate immunity to occur following sensitization with DNCB (8). Our data demonstrated that the increasecl functional status of peripheral bloocl granulocytes following chal- lenge with DNCB was accompanied by the increased survival of these cells. As terminally differentiated celi s, peripheral blood granulocytes die rapidly in cul ture (29), but their survival might be modulated in conditions of inflammation (30, 31), w here these cells are exposed to various inflammato1y mediators, including cytokines (32, 33). Skin sensitization with haptens was demon- strated to increase levels of circulating IL-6 (34) a cytokine, which migh t delay apoptosis of PMNs (30). Thus, the increased index of spontaneous MTT reduc- tion in cultures of granulocytes from challengecl rats, might result from a previous exposure of granulocytes to inflammato1y mecliators, including cytokines, in the circulation. Our data, which clemonstrated a prolonged peripheral blood granulocyte survival following sensi- tization of rats with DNCB, further increased following in vitro granulocyte incubation with autologous plasma, support this assumption (8). Overall , these findings support a role of granulo- cytes in the elicitation of CHS. Indeed, depletion of granulocytes before the elicitation prevented the ex- pression of CI-IS in mice (7). As changes in peripheral blood granulocyte activity were noted cluring the sen- sitization pluse also (8), these, along with the presented data strengthen a contribu to1y role of these cells in con- tact sensitivity. In this regard, a role for skin-infiltrating neutrophils in the recruitment of hapten-specific CD8+ T cells to the site of hapten challenge was suggested (7). Taken together, these data imply that experimental contact hypersensitivity might be viewed not only as a system for studying the role of hapten-specific but for non-specific mechanisms of this cutaneous response, as well. One further aspect of the rat CHS model should be mentioned. It is a frequently used experimental animal model in toxicity and immunotoxicity studies (14, 35, 37, 38) ancl in testing of chernicals and drugs to be ap- proved for use in humans (35 , 36). In this regard , study- ing hapten-specific as well as non-specific aspects of this cutaneous reaction following a xenobiotic expo- sure might give better insights into mechanisrns of im- rnune rnodulation by these agents. In surnrna1y, the contact hypersensitivity reaction induced by DNCB in rats represents, in our opinion, a suitable experirnental model for studying both antigen- specific and antigen-nonspecific mechanisms of cuta- neous irnrnune responses to contact sensitizing chemi- cals. It rnay prove to be very useful fo"r studying both local and systemic immunomodulato1y / immunotoxic effects of various chemicals and physical agents in vivo. Examination of these effects in the same animal model might represent a more integrated approach to the immunodermatotoxicological research. Acta Dermatoven APA Vol 10, 2001, No 3 - --------------------------- --- - - -- 89 Experimental contact sensitivity wul innate immunity lmmuno l o g i c study R_ E F E .H E N C E S l. Rheins A. What's new in cutaneous toxicity. J Toxicol Cut Ocular Toxicol 1992; 11: 225-38. 90 2. Lappin MB, Kimber I, Norval M. The role of dendritic cells in cutaneous immunity Arch Dermatol Res 1996; 288: 109-21. 3. Roupe G, Ridell B. 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Peptide immu- nization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity. J Invest Dermatol 2000; ll5: 260-6. 20. Bunce C, Bell EB. CD45 isoforms dcfine two types of CD4 memory T cells, one which depends on persisting antigen. J Exp Med 1997; 185: 767-76. 21. Scheynius A, Skoglund C, Johanson C, Holmdahl R. The ral as an experimcntal model for studies on immune regulating mechanisms in contact allergic reactions. Acta Denn Venereol (Suppl.) 1988; 135: 54-56. - ----- ----- --------------------- --Acta Dermatoven APA Vol 10, 2001, No 3 Immunolo gic stud y AUTHORS ' ADDRESSES Experimental contact sensitivity and innate immunity 22. Zhang L, Tinkle SS. Chemical activation of innate and specific immunity in contact dermatitis. J Invest Dermatol 2000; 115:168-76. 23. Kakinuma K, Fukuhara V, Kaneda M. The respiratory burst oxidase of neutrophils. J Biol Chem 1987; 262: 12316-24. 24. Hallet M, Lloyds D. Neutrophil priming: the cellular signals that say "amber" but not "green". Immunol Today 1995; 16: 264-8. 25. Forsyth K, Levinsky RJ. Role of the LFA-1 adhesion glycoprotein in neutrophil adhesion to endothe- lium and plastic surfaces. Ciin Exp Immunol 1989; 75: 265-8. 26. Arnaout A. Structme and function of the leukocyte adhesion molecules CD11/CD18. Blood 1990; 75: 1037-50. 27. Nakamura M, Rikimaru T, Yano T, Moore G, Pula PL, Shofield BH, Dannenberg AM. Full-thickness human skin explants for testing the toxicity of topically applied chemicals. J Invest Dermatol 1990; 95: 325-32. 28. Luster MI, Wilmer JI, Germolec DR, Spalding J, Yoshida T, Gaido K, Simeonova PP, Burleson FG, Bruccoleri A. Role of keratinocyte-derived cytokines in chemical toxicity. Toxicol Let! 1995; 82-83: 471-6. 29. Haslett C. Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulo- cytes. Ciin Sci 1992; 83: 639-48. 30. 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Gill TJ III, SmHh, GJ, Wissler RW, Kunz HW. The rat as an experimental animal. Science 1989; 245: 269-76. 37. Lauerma AI, Homey B, Vohr HW, Lee CH, Bloom E, Maibach HI. Effect of topical cis-urocanic acid on local lymph node activation during contact sensitization in mouse, rat and guinea-pig. Br J Dermatol 1996; 134: 904-9. 38. Nelson CJ, 1-Iow T, Lysle DT. Enhancement of the contact hypersensitivity reaction by acute morphine administration at the elicitation phase. Ciin Immunol 1999; 93: 176-83. Milena Kataranovski PhD, Projessor ojimmunobiology, Faculty oj Biology, University ojBelgrade, Studentska 4, and Senior Research associate, Institutejor Medical Research, Military MedicalAcademy, 17 Crnotravslw, 11002 Belgrade, Yugoslavia, e-mail: vmaimi@eunet.yu Lidija Kandolf Sekulovic MD, Department oj Dermatology, Military MedicalAcademy, 17 Crnotravska, 11002 Belgrade, Yugoslavia, e-mail: bdd_sld@yubc.net Dordije J(aradaglic MD, PhD, Projessor oj Dermatology, Department oj Dermatology, Military MedicalAcademy, 17 Crnotravska, 11002 Belgrade, Yugoslavia, e-mail: bdd_sld@yubc.net Acta Dermatoven APA Vol 10, 2001, No 3 - ----------------------- -------- 91