ATPase Langerhans cell staining: a technique allowing progression from light to electron microscope observation.

A technique which enables good visualization of the membranous ATPase activity of epidermal Langerhans cells is described. The method has the advantage of keeping intact most of the ultrastructural details. It may allow the observation, under pathologic conditions, of ultrastructural modifications in ATPase-negative Langerhans cells still recognizable by their Langerhans cell granules.

Induction of tolerance to poison ivy urushiol in the guinea pig by epicutaneous application of the structural analog 5-methyl-3-n-pentadecylcatechol.

Previous studies have established that epicutaneous application of 5-methyl-3-n-pentadecylcatechol (5-Me-PDC), a synthetic analog of a poison ivy urushiol component, leads to immune tolerance to 3-n-pentadecylcatechol (PDC) in mice. The induction of tolerance by 5-Me-PDC may be mediated by a protein conjugate formed via selective reaction of thiol nucleophiles present on the carrier macromolecule with the corresponding o-quinone derived from the parent catechol. In order to examine further the tolerogenic properties of 5-Me-PDC, we have extended our studies to the guinea pig, the generally accepted experimental species for the study of contact allergy. The results have established that specific immune tolerance to poison ivy urushiol is induced following 2 epicutaneous applications of the PDC analog. Furthermore, we were able to show that the treated animals remained tolerant for at least 6 weeks, a period of time comparable to that observed following the intravenous administration of the O,O-bis-acetyl derivative of PDC. The data point to the possibility of developing a therapeutically effective topical tolerogen for poison ivy contact dermatitis.

Induction of tolerance to urushiol by epicutaneous application of this hapten on dinitrofluorobenzene-treated skin.

The application of a sensitizing dose of urushiol on a dinitrofluorobenzene (DNFB)-treated skin area significantly diminished the intensity of the urushiol challenge test in guinea pigs. Furthermore, the animals which had been first exposed to urushiol through DNFB-treated skin failed to become sensitized in a second sensitization attempt even when painted on a previously untreated area. This tolerance is hapten-specific and may be reversed by treatment with cyclophosphamide (200 mg/kg) shortly before another contact sensitization attempt to urushiol. In a previous work, we have shown that most of the Langerhans cells present in the DNFB-treated skin area are ATPase-negative and that there exists a link between the membranous ATPase system and the formation of Langerhans cell granules. The latter seem to develop in the course of a mechanism of adsorptive pinocytosis during which ATPase activity "disappears." Thus we suggest that the "unavailability" of ATPase-negative Langerhans cells for adequate processing a second hapten may result from the incapacity of cells lacking their ATPase system to activate the intracellular events that depend on this system and that normally lead to sensitization.

Suppression of urushiol-induced delayed-type hypersensitivity responses in mice with serum IgG immunoglobulin from human hyposensitized donors.

Serum IgG immunoglobulin fractions from human subjects hyposensitized to poison ivy/oak by oral administration of urushiol suppressed the induction of delayed-type hypersensitivity (DTH) responses in mice to this hapten. This suppressive activity was hapten specific because it did not modify DTH responses to dinitrofluorobenzene (DNFB). Absorption of human serum with lymph node cells from urushiolsensitized but not DNFB-sensitized mice removed the suppressive activity, suggesting that anti-idiotypic antibodies reacting with T-cell receptors are involved.

Regulation of murine contact sensitivity to urushiol components by serum factors.

Mice epicutaneously painted with components of poison ivy urushiol oil exhibit contact sensitivity (as detected by ear swelling reactions) that persist for about 25 days. Sera taken from mice at times when the contact sensitization response is waning suppressed the induction of sensitization to 3-n-pentadecylcatechol (PDC), a urushiol component, in recipients. The suppressive serum factor was present in greatest amount 25 days after sensitization, but was no longer detectable 40 days post sensitization. Suppression was antigen-specific, absorbed out with PDC-immune, but not normal lymph node cells, and transferable with a single 0.6 ml dose 7 days prior to sensitization of recipients. Suppression was transferable by the purified IgG fraction of desensitized mice. Results indicate that contact sensitivity to urushiol in mice is regulated by serum factors.

ATPase and morphologic changes induced by UVB on Langerhans cells in guinea pigs.

We have devised, in guinea pigs, an improved ATPase technique which enables one to proceed from light to electron microscope study while preserving, on the ultrastructural level, the various membranous structures, in particular the Langerhans cell (LC) granules. Using this method, we have been able to confirm the action of acute, low-dose UVB on the surface enzymatic marker, ATPase. Moreover, this study has shown that the ATPase-negative LC contain abnormal LC granules or, more often, are deficient in LC granules. In a previous work, we have shown that, after epicutaneous application of a hapten, one successively observes an extensive adsorptive pinocytosis process, the disappearance of the membranous ATPase system, and the appearance of LC granules in the cytoplasm. Therefore we may suppose that, after UVB irradiation, the disappearance of the ATPase system and/or the possible alteration of the adsorptive pinocytosis process interrupts or alters the formation of LC granules. These successive events might play a vital role in the formation of the hapten--carrier protein-Ia antigen complex. In their absence in a large number of LC, following UV irradiation, epicutaneous application of a hapten would lead to the development of a state of immune tolerance.

Human epidermal Langerhans cells internalize by receptor-mediated endocytosis T6 (CD1 "NA1/34") surface antigen. Birbeck granules are involved in the intracellular traffic of the T6 antigen.

Using immunogold staining of a suspension of living human epidermal cells to identify the Langerhans cell membrane-associated antigen T6 (revealed by the monoclonal antibody BL6), we have observed internalization of T6 antigen in Langerhans cells. This phenomenon is at least partly due to receptor-mediated endocytosis involving coated pits, coated vesicles, endosomes, the smooth endoplasmic reticulum, and lysosomes. These ultrastructural results suggest that T6 antigen may be part of a receptor site. Following receptor-mediated endocytosis, the appearance in the cell center of the first labeled Birbeck granules suggests that Birbeck granules could represent T6 intracellular transport organelles carrying T6 from the central part of the cell to an unknown destination.

ATPase and morphologic changes in Langerhans cells induced by epicutaneous application of a sensitizing dose of DNFB.

We have previously described an ATPase Langerhans cell (LC) staining technique allowing progression from light to electron microscope observation. Using this technique we have studied, following epicutaneous application of a sensitizing dose of a hapten, 2,4-dinitro-1-fluorobenzene (DNFB), the fate of the epidermal LC located in the sensitization zone. We wanted to know, under the light microscope, if the density and/or morphology of the LC are modified by such a treatment and, under the electron microscope, what are the ultrastructural changes accompanying the possible light microscope modifications. Under the light microscope, the observation of LC during the 5 d necessary for the development of contact sensitivity to DNFB shows that their number drops in the course of the first 24 h to normalize again 3 d later. Under the electron microscope, observations over the first 24 h revealed that LC remained in the epidermis, but were ATPase-negative. The disappearance of the membrane ATPase activity took place while the LC presented an increased number of coated pits, coated vesicles, endosomes, and lysosome organelles which characterize, at the ultrastructural level, the process of receptor-mediated endocytosis (RME). Following RME, many Birbeck granules (BG) appeared in the cytoplasm. Thus, epicutaneous application of DNFB leads to an endocytic activation of LC. However, the ligand(s) and/or the cell-surface components, which probably internalize during the RME process, remain unknown.

Synthesis of alpha-methylene-gamma-butyrolactones: a structure-activity relationship study of their allergenic power.

Thirty-five alpha-methylene-gamma-butyrolactones have been prepared and their allergenic properties tested on the skin of guinea pigs experimentally sensitized to (a) alantolactone (1), (b) isoalantolactone (2), and (c) alpha-methylene-gamma-butyrolactone (3). The two first groups of animals cross-react to lactones containing 9 to 18 carbon atoms but not to smaller alpha-methylene-gamma-butyrolactones. Conversely, animals sensitized to alpha-methylene-gamma-butyrolactone react only with alpha-methylene-gamma-butyrolactones containing 6 and 7 carbon atoms. These results are discussed in relation with the allergic contact dermatitis mechanism.

Perfluorinated analogues of poison ivy allergens. Synthesis and skin tolerogenic activity in mice.

3-(Tridecafluoroundecyl)catechol (8) and 3-(nonafluoropentadecyl)catechol (9), perfluorinated analogues of pentadecylcatechol (PDC), a constituent of poison ivy, have been synthesized. These compounds were nonsensitizers in mice. Compounds 8 and 9, however, were elicitors of allergic contact dermatitis in PDC-sensitized animals. Moreover, compound 9 exhibited tolerogenic properties to sensitization by poison ivy allergens, i.e. mice pretreated with perfluorinated compounds could not be sensitized to PDC.

Stereospecificity of allergic contact dermatitis (ACD) to enantiomers. Part III. experimentally induced ACD to a natural sesquiterpene dialdehyde, polygodial in guinea pigs.

Guinea pigs were sensitized to polygodial 1, a natural sesquiterpene dialdehyde, using intradermal injections in Freund's complete adjuvant. Cross-reactions with a (+/-)-mixture of warburganal and (-)-warburganal 2 (a natural hydroxylated derivative of polygodial) showed a high specificity of the allergic response since the (-)-pseudoenantiomer (having the same configuration as the primary sensitizer) gave a stronger skin reaction than the racemic mixture.

Allergic contact dermatitis to tulips: an example of enantiospecificity.

Allergic contact dermatitis (ACD), an immunological reaction of the skin resulting from contact with reactive compounds occurring in plants was shown to the enantiospecific (animals sensitized to a compound do not react to its nonsuperimposable mirror image). Thus, when guinea pigs were experimentally sensitized to (+)-tulipalin B (a compound present in tulip bulbs) they did not react to its enantiomer, (-)-tulipalin B. This was also true for (+)- and (-)-beta-hydroxy-gamma-methyl-alpha-methylene-gamma-butyrolactones.

Allergic contact dermatitis to laurel (Laurus nobilis L.): isolation and identification of haptens.

Two methods, using methylenelactone dimethylamino adducts, were used to remove selectively alpha-methylene gamma-butyrolactones from Laurus nobilis L. extracts. Isolated lactones were identified and "treated" extracts recovered. Two guinea-pig groups were sensitized to crude extracts and "treated" extracts, respectively, and tested with primary sensitizer and with different lactones. Only the first group showed strong skin reactions to crude extracts and to the lactones. Treated extracts were shown to be anallergic.

Allergic contact dermatitis to methylenelactones. Use of lymphocyte transformation test.

Seventeen guinea pigs were sensitized to alantolactone, a natural sesquiterpene lactone known for its sensitizing properties, using intradermal injections in Freund's complete adjuvant. Guinea pig skin protein extracts (SPE) were used to make conjugates with alantolactone and an isomer, isoalantolactone. Lymphocyte blastogenesis was observed with SPE-alantolactone conjugates, as well as with guinea pig albumin-lactone conjugates. In six cases, stimulation was noted with unconjugated haptens. These results do not show a high degree of hapten-carrier specificity in contact sensitivity to alantolactone, induced by unconjugated hapten injections. Cross-sensitivity with SPE-isoalantolactone conjugate was also observed.

Enantiospecificity in allergic contact dermatitis. A review and new results in Frullania-sensitive patients.

Allergic contact dermatitis is essentially enantiospecific: sensitization to one enantiomer generally does not imply elicitation to the mirror-image allergens. Examples from patients allergic to Frullania, and from the literature (usnic acids, 4-methoxydalbergiones, gamma-methyl-alpha-methylene-gamma-butyrolactones, frullanolides) are discussed.

Synthesis of and allergic contact dermatitis to bicyclo-[2,2,1]-heptyl-alpha-methylene-gamma-butyrolactones derived from norbornene and camphene.

Three new lactones, 2-oxo-3-methylene-4,7-methanobenzofuran and two alpha-methylene spirolactones, 3',3'-dimethylspiro(2-oxo-3-methylenefuran-5,2'-norbornane), were synthesized and their skin activity was tested on one human volunteer and on guinea pigs. The man, sensitized to Frullania, was found sensitive to the above lactones and also reacted to frullanolide and alantolactone. The guinea pigs, experimentally sensitized to alantolactone, cross-reacted to 2-oxo-3-methylene-4,7-methanobenzofuran and the two alpha-methylene spirolactones, and also to isolantolactone and frullanolide.

Patch testing with hexavalent chromium salts in different vehicles and with nickel and cobalt in petrolatum.

In approaching the question as to which test preparations are most suitable for patch testing of metals, 25 chromium sensitive patients were tested with 0.5% potassium dichromate either in water or petrolatum. The aqueous solution proved better than the petrolatum preparation at a 48 h reading with Neodermtest Roc. No difference in sensitivity was recorded between 4 hexavalent chromium salts. 3 patients who were only sensitive to chromium solution in water also reacted positively to a 1% cobalt chloride solution and a 5% nickel sulfate solution in petrolatum.

Cross-reaction in allergic contact dermatitis from alpha-methylene-gamma-butyrolactones: importance of the cis or trans ring junction.

Cross-reaction in allergic contact dermatitis (ACD) is a highly stereoselective process. The importance of the cis or trans ring junction in alpha-methylene-gamma-butyrolactones in cross-reactivity was investigated by reacting Helenin (mostly a mixture of natural allergenic sesquiterpene lactones alantolactone 1 and isoalantolactone 2, which present a cis ring junction) in guinea pigs sensitized to model allergenic alpha-methylene-gamma-butyrolactones: cis-bicyclic lactone 3 and trans-bicyclic lactone 4.