Route of contact sensitization and in vitro lymphocyte transformation

A guinea pig skin extract conjugate with dinitrofluorobenzene elicited significant in vitro transformation of cultured lymphnode lymphocytes from 19 of 27 guinea pigs sensitized by footpad injection of dinitrochlorobenzene in Freund's complete adjuvant, as compared to only 1 of 26 guinea pigs topically sensitized to dinitrochlorobenzene. Topically sensitized guinea pigs appear to be more appropriate models for contact allergy in man than guinea pigs sensitized by other methods. Other sensitization procedures are likely to produce more heterogeneous forms of sensitization, with features of contact allergy, tuberculin-type allergy, antibody-mediated hypersensitivity and cutaneous basophile hypersensitivity.     

Relationship between macrophage infiltration and epidermopoiesis in delayed-type hypersensitivity

Summary The relationship between epidermopoiesis and macrophage infiltration was studied in delayed-type hypersensitivity (DTH) skin lesions in guinea pigs that had been sensitized with heat-killed tubercle bacilli or bovine serum albumin (BSA). Macrophages were identified with acid-phosphatase and nonspecific esterase stains, and the epidermal proliferative response was studied at DTH challenge sites by autoradiography. The number of macrophages in the sensitized animals was higher than that in the nonsensitized animals 48–72 h following challenge injections, when labelling indices were also elevated in the former group. Soluble factor(s) from cultured macrophages transiently enhanced the DNA synthesis of epidermal cells in cultures and in the sites injected with the factor(s). These results suggest that macrophages retained in the DTH lesion may play a role in an acceleration of epidermal proliferation, thus leading to acanthosis and lichenification.     

Sodium dodecyl sulfate induces plasminogen activator inhibitor type 2 expression in epidermal keratinocytes in vivo and in vitro

The detergent sodium dodecyl sulfate is a well-known inducer of irritant contact dermatitis. In this study we show that sodium dodecyl sulfate induces the serine proteinase inhibitor, plasminogen activator inhibitor type 2, in epidermal keratinocytes. The enhancement in plasminogen activator inhibitor type 2 mRNA and antigen is observed both when sodium dodecyl sulfate is applied topically to normal human skin as well as when it is added to the growth medium of cultured human keratinocytes. In vitro, plasminogen activator inhibitor type 2 mRNA is increased within 4-8 h after addition of the detergent, and the increase in plasminogen activator inhibitor type 2 antigen occurs slightly later. The enhancing effect of sodium dodecyl sulfate on plasminogen activator inhibitor type 2 is not related to nonspecific cell lysis nor is it secondary to induction of tumor necrosis factor alpha. Similarities between our in vitro and in vivo findings lead us to hypothesize that sodium dodecyl sulfate may exert its effect on epidermal plasminogen activator inhibitor type 2 via interaction with the keratinocyte.     

The mechanisms of histological incontinence of pigment: light and electron microscopic studies of lesions and patch test sites in female facial melanosis and DNCB reactions in guinea pigs

Histological incontinence of pigment (HIP) was studied using light and electron microscopy in a pigmented lesion from a female facial melanosis (FFM) patient and in another lesion produced by isoeugenol when the patient was patch tested. The following sequence of events is thought to underlie HIP. Phagocytes invade the epidermis and phagocytize melanosomes either in keratinocytes or in cytoid bodies, which are degenerated keratinocytes. These phagocytes then return to the dermis through gaps in the basal lamina. In brown guinea pigs sensitized to DNCB, 0.02% DNCB in acetone was applied repeatedly to the same area of the abdomen once per week for six weeks. HIP was observed in 28% of the animals following the fifth application of DNCB. In contrast to the HIP process observed in the patient, phagocytes in brown guinea pigs phagocytized free melanosomes. In irritant reactions to DNCB in brown guinea pigs, only concentrations which produced epidermal necrosis induced HIP.     

Langerhans'' cell population in topical patch-test and systemic flare-up sites of nickel-sensitive guinea pigs

Guinea pigs were sensitized to nickel by using the maximization method of Magnusson and Kligman (1969), challenged by topical and systemic applications of the allergen, and the epidermal Langerhans' cell (LC) density studied with ATPase histochemistry. Similar LC counts were found in unchallenged skin of nickel-sensitized and control animals (657 ± 151 vs. 671 ± 16 cells/mm²). Topical patch-test challenge with nickel sulphate lowered the LC count in both sensitized and control animals to some extent, but the changes were not statistically significant. In contrast, systemic (intraperitoneal) nickel sulphate challenge induced a statistically significant (P <0–001) LC decrease in the previous topical test sites of sensitized guinea pigs, while no such decrease was evident in the corresponding skin sites of control animals. The method of nickel sensitization, topical challenge, and subsequent systemic challenge is a versatile experimental model for studying the pathology of the LC population in delayed-type hypersensitivity skin reactions.     

Epidermal hyperplasia induced in guinea pig flank skin by intradermal injection of Sudan red.

Studies of dermal-epidermal interactions were conducted with guinea pig flank skin and intradermal injections of the irritant, Sudan IV dye in olive oil. These injections led to epidermal hyperplasia in areas overlying the irritant and the effect was most significant when the irritant was placed in the upper dermis. Basal cell mitotic activity and thymidine uptake reached a peak by 24 h and thereafter dropped rapidly. Maximal epidermal thickness (4.3 times the control) resulting from an increase in cell number occurred within 2-4 days. Despite the very short period of increased cell growth, epidermal thickness returned to control values only after a 24-day period. A similar growth response could not be induced by saline injections. A single topical application of the irritant showed a qualitatively and quantitatively different epidermal response. These experiments indicate that an intradermal irritant can lead to epidermal hyperplasia and a long-lasting epidermal thickening.     

Mediators, initiating the inflammatory response, released in organ culture by full-thickness human skin explants exposed to the irritant, sulfur mustard.

Mediators released from injured human skin that initiate the inflammatory response have not been adequately identified. Organ culture of full-thickness skin explants enables us to do so, because injury to the skin can be made in vitro, eliminating the rapid leakage of serum and infiltration of leukocytes that occur in vivo. In our studies, the military vesicant sulfur mustard (SM) (10 microliters of a 0.01 to 1.0% dilution) was topically applied to injure the epidermis of the explant. Then, the explants were cultured in small Petri dishes, usually for 18 h at 36 degrees C, and the organ-culture fluids were assayed for various inflammatory mediators. We found that the culture fluids from SM-exposed and control explants contained similar amounts of angiotensin-converting enzyme, trypsin-like and chymotrypsin-like proteases, acid phosphatase, beta-glucuronidase, beta-galactosidase, lysozyme, deoxyribonuclease, ribonuclease, interleukin 1, and lactic dehydrogenase. However, the culture fluids from SM-exposed explants contained increased amounts of histamine and plasminogen-activating activity, and often prostaglandin E2, when compared to culture fluids from control explants. After 3 to 4 d in culture, full-thickness human skin explants, when exposed to 0.2% SM (but not when exposed to 1.0% SM), sometimes showed separation of the epidermis and increased collagenase activity (i.e., hydroxyproline release). Thus, histamine (from local mast cells), and prostaglandin E2 and plasminogen-activating activity (probably from both mast cells and epidermal cells) are apparently involved in early mediation of the inflammatory response.     

Value of the cutaneous basophil hypersensitivity (CBH) response for distinguishing weak contact sensitization from irritation reactions in the guinea pig

Numerous studies of the histology of allergic contact dermatitis reactions to potent allergens in guinea pigs and humans have indicated that there is significant tissue infiltration with basophilic leukocytes. In this study we determined whether this histologic finding could be of value in distinguishing weak sensitization reactions from primary irritation, thereby aiding in the predictive identification of weak or moderate contact allergens. Guinea pigs were sensitized by the Buehler test method. Skin reactions were graded 24, 48, and 72 h post-challenge with duplicate patch sites biopsied at the 24- or 72-h grading timepoints. The biopsies were fixed, embedded in glycol methacrylate, thin sectioned, and Giemsa stained. The number of basophils per 400 leukocytes were counted along the upper dermis just below the dermal/epidermal junction. Challenge patch sites from animals sensitized to a relatively low dose of the strong contact allergen, oxazolone, were compared with patch sites from animals challenged only with a strong irritant, sodium lauryl sulfate (SLS). Compared to normal skin (7.5 +/- 1.0 basophils/400 leukocytes +/- SEM) only the oxazolone patch sites showed significant basophil infiltration (36.8 +/- 6.5), despite the fact that the skin reactions to the low oxazolone challenge dose were relatively weak. SLS patch sites showed no basophil infiltration above normal skin levels (4.8 +/- 0.9). Subsequent blinded studies compared weak/moderate presumptive sensitization reactions (as defined by accepted visual skin grading criteria) to various chemicals (citronellal, vanillin, cinnamic aldehyde, and ethylenediamine) to primary irritation reactions to the same chemicals. In each case, low-challenge-dose sensitization sites on previously treated (induced) animals showed mean basophil infiltration (range, 11.9-69.2 basophils/400 leukocytes) significantly greater than higher-dose irritant reactions (range, 1.6-13.3). The range for normal skin was 0.2-10.2 and the range for strong patch reactions to higher concentrations of oxazolone was 59.8-209.3. These data strongly indicate that light-microscopic quantitation of the CBH response can be used to distinguish relatively weak to moderate contact sensitization reactions from primary irritation reactions to the same chemicals.     

Histochemical analysis of Langerhans' cells.

Single cell suspensions of epidermal cells from guinea pigs were analyzed histochemically for the presence of the following enzymes: 5'-adenosine triphosphatase, nonspecific esterase, specific esterase, myeloperoxidase and leukocyte alkaline phosphatase. A population of cells was positive for nonspecific esterase, leukocyte alkaline phosphatase, and 5'-adenosine triphosphatase. This population was identified as Langerhans' cells because the number of cells stained for these enzymes paralleled the number of Langerhans' cells in the suspension. These same enzymes were shown to be present in guinea pig leukocytes of the mononuclear-phagocytic series, suggesting that they and Langerhans' cells may have a precursor in common.     

Lymphocyte transformation to membrane-conjugated,liposome-conjugated,or unconjugated pentadecylcatechol in the guinea pig

Summary This study examined the in vitro immunogenicity of haptenated liposomes and compared them with haptenated biological membranes and unconjugated hapten. Peripheral-blood lymphocytes were obtained from guinea pigs topically sensitized with pentadecylcatechol (PDC) and immunologically naive guinea pigs. Lymphocyte transformations were studied by [³H]thymidine uptake. PDC failed to stimulate the lymphocytes from the immunologically naive group. There was significant blastogenesis in the cells of the sensitized group, but the degree of stimulation was dependent upon the manner in which the antigen was presented to them. Unconjugated hapten caused a low-level, dose-dependent mitogenesis in the sensitized T cells and hapten-conjugated liposomes enhanced this response (P<0.05). By far the most effective immunogen was a haptenated biologic membrane. In all cases, the mitogenic response was macrophage dependent. It is possible that the haptenated biologic membranes were more effective than synthetic membranes (liposomes) because of the presence of membrane proteins that can conjugate with hapten and from a more effective immunogen.     

Epidermal cell proliferation following an active arthus reaction in the guinea pig

Active Arthus reactions were provoked by injections of 100 micrograms horseradish peroxidase (HRP), 10 micrograms HRP and 100 micrograms bovine serum albumin (BSA) into the skin of sensitized guinea pigs. Labeling indices (LI) of epidermal basal cells were measured 1, 4, 8, 24, 48 and 72 h later by the in vivo 3H-thymidine labeling technique, and compared with those obtained with injections of antigens into the skin of non-sensitized guinea pigs. From 1-8 h after the induction of an active Arthus reaction, the LI of epidermal basal cells of the skin injected with 100 micrograms HRP decreased to a remarkably low value. On the other hand, those obtained with the reaction against 10 micrograms HRP were significantly high. At 24 h after the reaction, LI were as high as those obtained in non-sensitized guinea pigs with control intradermal injections, though the former persisted high until 48 h after the injection. In addition, decreased LI of the epidermal basal cells were observed in the skin 4 h after intradermal injections of immune complexes. It was suggested that DNA synthetic activity of the epidermis increases in a mild active Arthus reaction, while the activity may be suppressed in a severe active Arthus reaction up to 8 h after provocation.     

Animal model for assessment of skin irritancy

Irritant skin reactions from repeated open applications of low concentrations of sodium lauryl sulphate (SLS) have been studied macroscopically and microscopically in guinea pigs. After 3 applications daily for 3 days, 2% SLS aqueous solutions gave a naked eye assessment, increase in epidermal thickness and total dermal inflammatory cell response, which was greater than for a 1% SLS solution. The dermal inflammatory cell response was mainly mononuclear (lymphocytic) in nature. With the SLS reactions as control, various organic solvents were studied and ranked against the SLS reactions and internally. Trichlorethylene was the most irritant solvent, ranking as high as 2% SLS. Other chlorinated hydrocarbons and aromatic hydrocarbons tested caused irritant reactions. The alcohols and acetone gave no reaction. White spirit was as irritant as trichlorethylene. Thinners were less irritant, around the level of the 1% SLS control reaction. The 4-day experimental design is a convenient and suitable animal model for screening irritant potential, and provides information relevant to the pathogenesis of irritant contact reactions.     

中药槲皮素对皮肤增色作用的动物实验研究

目的研究槲皮素对豚鼠表皮中黑素细胞的影响,观察外用槲皮素对实验动物的安全性和疗效,为白癜风等色素减退性皮肤病的中药治疗提供实验依据。方法用槲皮素乙醇提取液外涂豚鼠背部皮肤并予红外线照射,5周后对涂药皮肤和对照皮肤进行活检。用不同的染色方法分别制备病理切片,观察黑素细胞形态学改变,统计给药后表皮病理切片中黑素细胞数目和黑素含量。结果豚鼠表皮外用槲皮素后黑素细胞数目明显增加,槲皮素实验组多巴阳性黑素细胞数及含黑素颗粒细胞数均较空白对照组显著增多,与阳性对照组8-甲氧补骨脂素(8-MOP)差异无显著性。结论槲皮素对棕黄色豚鼠背部皮肤有增色作用,具有促进黑素细胞增殖和黑素合成的功能。     

A new animal model for contact dermatitis: the hairless guinea pig.

Allergic and irritant contact reactions were evaluated in the recently identified hairless guinea pig, Crl:IAF(HA)BR, a mutant from the Hartley strain. The cutaneous changes were observed macro- and microscopically. The irritant contact dermatitis was induced by croton oil, 2,4-dinitrochlorobenzene (DNCB), or anthralin. Both hairless and hairy guinea pigs developed similar reactions to these chemicals. The density of the epidermal Langerhans cells (LC) of hairless guinea pigs was significantly higher than that in the hairy strain. Allergic contact sensitization was easily induced with DNCB. Photoallergic contact sensitization was also induced with tetrachlorosalicylanilide (TCSA) but not with tribromosalicylanilide (TBS). However, by administration of cyclophosphamide before sensitization, positive photocontact responses were seen with TBS. These results indicate that hairless guinea pigs can be used as animal models for investigation of immunologic and nonimmunologic contact reactions.     

The pathogenic mechanisms of blister formation in bullous pemphigoid

Normal human skin was cultured with sera, IgG fractions, and blister fluids (BF) from patients with bullous pemphigoid. Antibody binding (IgG) was observed by immunofluorescence techniques at the dermal-epidermal junction of all skin explants culture with sera, IgG fractions, and BF. Dermal-epidermal separation was observed only in the skin explants cultured with BF. Dermal-epidermal separation was observed in 19 out of 20 explants cultured with BF obtained from fresh bullae. In addition, dermal-epidermal separation can be produced in vivo 6 hr after the injection of BF into the dorsal skin of Hartley guinea pigs. Dermal-epidermal separation was not observed in skin explants cultured with heat-inactivated (56 degrees, C 30 min) BF, although antibody binding was observed. In addition, dermal-epidermal separation did not occur when the BF were preincubated with rabbit antihuman C1, C3, C4, and C5 antibodies. These observations suggested that both antibody and complement were essential for the production of dermal-epidermal separation. Since patient sera failed to produce dermal-epidermal separation, other factor(s) present in BF but absent from serum might be necessary for the production of dermal-epidermal separation. The addition of the proteinase inhibitors, pepstatin, EDTA, and soy bean trypsin inhibitor, did not inhibit the formation of dermal-epidermal separation. In contrast, the presence of alpha 2-macroglobulin inhibited dermal-epidermal separation.     

Sensitization of mice to paraphenylenediamine and structurally-related compounds: adjuvant effects of vitamin A supplementation

Allergic contact dermatitis from moderate and weak contact sensitizers is generally studied with guinea pigs, since they are readily sensitized to contact allergens. Mice, by contrast, are poor responders to weak contact allergens. However, the variety of in vitro murine systems us well, is murine specific reagents make mice the preferable species, with the use of vitamin A supplementation, 2 protocols were developed which sensitized CBA/J female mice to paraphenylenediamine, Mice were sensitized by 5 daily topical applications to shasen dorsal skin. Alternately mice were sensitized by 2 intraperitoneal infections of antigen pulsed spleen cells. Sensitization to paraphenylenediamine was determined by ear swelling following topical application. Vitamin A supplementation was found to be essential for optimum response. Lymph node and spleen cell from sensitized mice were capable of proliferating to paraphenylenediamine in vitro. With the use of Vitamin A supplementation and intraperitoneal injection, CBA/J mice were also sensitized to a number of compounds structurally related to paraphenylenediamine, including the ortho- and meta-derivatives of paraphenylenediamine, as well as hydroquinone and resorcinol. These new protocols, combined with vitamin A supplementation, expand the use of mice to study moderate sensitizers with minimal animal utilization.     

Optimization of an in vitro lymphocyte blastogenesis assay for predictive assessment of immunologic responsiveness to contact sensitizers

Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.     

Sequence of morphological events during topical application of retinoic acid on the rhino mouse skin

In a study of the comedolytic action of retinoids on the epidermal pseudocomedones of rhino mouse skin, the earliest changes and the sequence of events induced by the treatment were investigated. Rhino mice were treated topically with all-trans retinoic acid and at various time intervals from day 0 to 3 weeks, histological and ultrastructural studies as well as quantification of mast cells were performed. The earliest changes occurred in the dermis after 2 days of treatment and were characterized by degranulation of mast cells, clumping and association of Langerhans cells and lymphocytes. During the second week of treatment there was hyperplasia, hypergranulosis and an increase in the number of membrane coating granules with disorganization of the horny layer both of the epidermis and the follicular epithelium. These changes in the proliferation and the differentiation of keratinocytes resulted in a comedolytic effect. Associated with these epidermal changes there were changes in the dermis with an increased number of mast cells and a decreased number of Langerhans cells.     

Effect of indomethacin on alteration of ATPase-positive Langerhans cell density and cutaneous sunburn reaction induced by ultraviolet-B radiation

We have investigated the effect of ultraviolet-B (UVB) irradiation on the density of epidermal ATPase-positive Langerhans cells, and the modulation of this effect by indomethacin (IND). Depilated backs of albino guinea pigs were exposed to varying doses of UVB (10-550 mJ/cm2). Skin biopsies were taken serially. There was an UVB dose-dependent decrease in the density of dendritic epidermal Langerhans cells, as identified by their membrane ATPase activity. This was accompanied by thinning and shortening, or disappearance of dendritic processes. Such changes were followed by a gradual recovery of the cell density to preirradiation level by day 21. Despite the high doses of UVB given, the maximal decrease in the density of ATPase-positive cells was only 58%. Topical application of IND, a prostaglandin-synthetase inhibitor, after irradiation resulted in a decrease of the erythema; however, the decrease in the density of ATPase-positive cells was still observed. In contrast, guinea pigs that received IND topically prior to irradiation showed a decrease erythemal response, but failed to show any decrease in the density of ATPase-positive cells. Administration of IND orally for 3 days prior to UVB exposure did not prevent the decrease in the cell density. The protective effect of topical IND, applied prior to irradiation, may be explained by its in vitro absorbance at both the UVB and UVA ranges. Topical application of IND 20 min prior to exposure to UVB in 2 human subjects resulted in an increase in the minimal erythema dose, giving a sun protection factor of 1.6, which is comparable to that produced by an equimolar concentration of para-aminobenzoic acid solution. The sun-protective property of IND, together with its activity as a prostaglandin synthetase inhibitor, indicate that it potentially could be a useful sunscreen agent. Its clinical safety and efficacy, however, remain to be determined.     

Patch testing with beryllium alloy samples in guinea pigs

An experimental study was conducted in guinea pigs for the predictive assessment of the beryllium alloy hazard in occupational exposure of the skin to beryllium compounds. Guinea pigs were sensitized to beryllium sulfate according to the maximized Magnusson and Kligman test, and challenged with beryllium alloys and metallic copper, beryllium and aluminum samples. Results showed a delayed skin hypersensitivity reaction in 30 to 60% of pre-sensitized guinea pigs challenged with copper-beryllium alloys and aluminum-beryllium alloy. An inflammatory follicular reaction was induced by copper in both controls and pre-sensitized guinea pigs.