8 week-old female BALB/c mice were purchased from Envigo, Netherlands. The Local Ethics Committee of the Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland gave consent # 039/2022 to perform the experiments. The mice were housed in cages at 21–22 °C with a 12/12-h light/dark cycle and were fed a commercial, pellet food as well as water ad libitum.

Hanks’ balanced salt solution (HBSS, L0606), phosphate buffered saline (PBS, L0615) were from Biowest (Nuaillé, France), bovine serum albumin (BSA, A7030), oxazolone (OXA, E0753) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, M2128) were from Sigma-Aldrich (St. Louis, MO, USA), May-Grünwald (1030.2) and Giemsa stains (1020.2) from Aqua-Med, Łódź, Poland. The reagents used in the histological and histochemical investigations were as follows: formalin (114321729), xylene (115208603) from Chempur, Piekary śląskie, Poland; hematoxylin (497-03971), eosin (17372-87-1), toluidine blue (89640), Meyer’s hematoxylin (MHS16) from Merck KGaA, Darmstadt, Germany; Ly6G antibody (clone RB6-8C5, MA1-10401), T lymphocytes antibody (clone SP7, MA5-14524) from Thermo Fisher Scientific, Waltham, MA, USA; Langerin/CD207 antibody (clone 929F3.01, DDX0362P-100) from Novus Biologicals, Littleton, CO, USA; ImmPRESS Reagent Kit anti-rabbit Ig (MP-7451) and ImmPRESS Reagent Kit anti-rat IgG (MP-7444), chromogens: ImmPACT™ DAB (SK-4105) and ImmPACT™ NovaRED™ (SK-4805) from Vector Laboratories Inc, Newark, CA, USA.

Yolkin was prepared from hen egg yolks as previously described [30]. The yolkin preparation contained three main proteins with a molecular weight below 45 kDa.

The mice were divided into four groups: (1) control, unstressed mice (Ctrl), (2) unstressed mice drinking yolkin solution in tap water (Yolkin), (3) stressed mice (Ctrl Stress) and (4) stressed mice drinking yolkin (Yolkin/Stress). The control, stressed mice underwent stress immobilization, i.e., they were kept in 50 mL perforated tubes, 4 h daily, for 5 consecutive days and had access during period 20 h/day to standard filtered tap water. Other stressed mice had access during this period to filtered tap water containing 5 µg/mL of yolkin. Unstressed mice from the Ctrl and Yolkin groups during the stress period remained in their cage, but similarly to stressed mice, without access to water and food for 4 h daily. 24 h later the mice were administered buprenorphine and the auricle edema was measured. Then, the mice were anaesthetized with 3% of isoflurane, bled and sacrificed by cervical dislocation. The auricles were cut off for histological examinations and thymocytes and splenocytes were isolated for immunological analysis. The experimental design is presented in Fig. 1.

Fig. 1.

The experimental design. OXA, oxazolone.

After buprenorphine (Bupaq Multidose) administration and measurement of the ear thickness, the mice were anaesthetized with isoflurane (Isoflurin, Vetpharma Animal Health, SL, Barcelona, Spain) bled and sacrificed by cervical dislocation. The thymuses and auricular lymph nodes were isolated and disintegrated to single cell suspension in cold PBS, using syringe piston and plastic screen.

The splenocytes were obtained as above using 0.83% NH₄Cl solution, instead of PBS, to lyze erythrocytes for 5 min incubation at room temperature (RT). The cells were subsequently washed twice with Hanks’ solution, passed through cotton wool columns to dispose debris, and re-suspended in the culture medium consisting of RPMI-1640 (Roswell Park Memorial Institute 1640 medium, L0500, Biowest, Nuaillé, France), supplemented with 10% fetal calf serum (FCS, S181B, Biowest, Nuaillé, France), sodium pyruvate (L0642, Biowest, Nuaillé, France), 2-mercaptoethanol (M3148, Sigma-Aldrich, St. Louis, MO, USA) and antibiotics (L0022, Biowest Nuaillé, France). For enumeration of cells in the lymphoid organs Türk’s solution (109277, Sigma-Aldrich, St. Louis, MO, USA) was used. The numbers of viable cells were counted in a hemocytometer using 0.1% Trypan blue reagent (T6146, Sigma-Aldrich, St. Louis, MO, USA) in PBS.

The pooled splenocytes (5 $\times$ 10⁶ cells/mL/well in 24-well plates), cultured in the medium of the composition described above, derived from the organs of mice on the day of elicitation of CS. The cultures were treated for 44 h with 2.5 µg/mL of ConA or 1 µg/mL of LPS (C7275 or L4130 respectively, Sigma-Aldrich, St. Louis, MO, USA). The contents of interferon gamma (IFN $\gamma$) and IL-6 in the culture supernatants were measured using commercial kits (430815 and 431315 respectively, Biolegend, San Diego, CA, USA). To determine cell proliferation, the splenocyte cultures were distributed into 96-well plates in 100 µL aliquots (2 $\times$ 10⁵ cells/well). ConA (2.5 µg/mL) or LPS (25 µg/mL) or were applied to induce T and B cell proliferation, respectively. After 72 h incubation with LPS or 48 h culture with concanavalin A (Con A), the cell proliferation was determined by the colorimetric MTT method.

Circulating blood from the retroorbital plexus was obtained in isoflurane anesthesia on the day of elicitation of contact sensitivity. The sera were isolated and kept at –80 °C until corticosterone determination using a Demeditec Diagnostics GmbH kit (DEV9922, Kiel, German).

The originally described test [33] was used with some modifications. 100 µL of 0.5% OXA dissolved in acetone was applied on the shaved abdomen. After 5 days, 50 µL of 1% OXA was administered to both sides of the auricles. The ear thickness was measured by means of a spring caliper after 24 h following application of the second dose of antigen. The results were shown as an antigen-specific increase of the ear thickness, i.e., background (BG) values (mice given only the eliciting dose of the antigen) were subtracted.

The paraffin auricle sections were prepared in a standard procedure and stained with hematoxylin and eosin (H&E). The staining of cells with respective antibodies was performed according to the manufacturers’ instructions (Immunohistochemistry kits; ImmPRESS Reagent Kit anti-rabbit Ig (MP-7451) and ImmPRESS Reagent Kit anti-rat IgG (MP-7444), Vector Laboratories Inc, Newark, CA, USA). Cells were counted in 3 random fields for each animal. In the morphometric analysis a cross-sectional area of the pustules in the epidermis (expressed in µm²) was measured. Quantitative and morphometric measurements of pustules were performed on both sides of the auricles, from the base toward the apex. Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), with imagine software NIS Elements (Nikon Corporation Tokyo, Japan), at 100$\times$ magnification was used for the analyses.

The calculations and statistical analysis were accomplished using Microsoft® Excel XP (Microsoft, Warszawa, Poland) and STATISTICA® 7.0 programs for Windows (StatSoft Polska, Kraków, Poland). Eight mice per group were used in this study. The statistical significance of differences between the mean values was tested by means of variance analysis (ANOVA) for the one-factor classification with post hoc analysis (multi comparisons) by RIR Tukey’s test. The evaluation of normality of the variable distribution was performed with the W Shapiro-Wilk test, and the evaluation of the homogenous variance assumption with Brown-Forsyth’s test. Moreover, the existence of dependencies between group means and variances were verified. Fulfillment of the above assumptions (normal or symmetric distribution of variables, homogeneity of variance, lack of dependencies between means and variances) made it possible to use the variance analysis for testing the mean equality hypothesis. If these assumptions were not fulfilled, a non-symmetrical Kruskal-Wallis’ test was used. The significance was set at p 0.05.

The mice were subjected to immobilization stress for 5 days followed by immunization with OXA. One group of mice was administered yolkin in their drinking water throughout the stress period. Fig. 2 shows the results of the measurement of the auricle edema after 24 h following elicitation of the contact sensitivity reaction. Yolkin alone had no effect on the magnitude of the CS reaction, but the psychic stress led to a significant (more than 50%) decrease of the ear edema. Although the treatment with yolkin did not completely restore the CS response to control levels, its reconstituting effect on the ear edema was statistically significant.

Fig. 2.

The effects of stress and yolkin on the antigen specific thickness of auricles of mice with fully developed CS to oxazolone. The exposure of mice to stress and the performance of the contact sensitivity test are described in sections 2.4 and 2.8. The experimental design is presented in the Materials and Methods section. Briefly, mice drank water containing yolkin (5 µg/mL) for 5 days during stress. Next day 0.5% OXA was applied on the skin of their abdomens. The CS effectual reaction was evoked after 5 days by application of 1% OXA on both sides of auricles. The thickness of auricles was determined after 24 h with a spring caliper. The antigen specific increase of ear thickness (BG values are subtracted from the responses of sensitized mice) is presented as a mean value of ear thickness measured in 8 mice (16 determinations) in mm $\pm$SD. *** statistically significant (p 0.001). CS, contact sensitivity; OXA, oxazolone; BG, background.

As shown in Fig. 3A, at the time of elicitation of CS, the IFN $\gamma$ levels in the splenocyte cultures stimulated with Con A were comparable in control and yolkin-treated mice. The splenocytes derived from the stressed mice produced less IFN $\gamma$ (a statistically significant decrease). This effect was, however, reversed in the stressed mice drinking the yolkin solution.

Fig. 3.

The ex vivo effects of oral yolkin administration during restraint stress and development of contact sensitivity on Con A- and LPS-induced IFN $\gamma$ production by splenocyte cultures. The treatment of mice with yolkin and determination of the CS is described in the legend to Fig. 2. The experimental design is presented in the Materials and Methods section. The pooled splenocytes from four mice were isolated 24 h after elicitation of the CS. (A) Stimulation by Con A. (B) Stimulation by LPS. The results are presented as mean values from triplicate measurements in pg/mL $\pm$ SD. ** and *** statistically significant (p 0.01 and p 0.001, respectively). Con A, concanavalin A; LPS, lipopolysaccharide; IFN $\gamma$, interferon gamma.

Similar results were obtained in the splenocyte cultures stimulated with LPS (Fig. 3B) although the upregulatory effect of yolkin on IFN $\gamma$ production in the splenocytes from stressed mice was stronger.

A similar inhibitory effect of stress was observed in IL-6 production by Con A-induced splenocytes (Fig. 4). The effects of yolkin alone or stress were not significantly inhibitory. On the other hand, the treatment of the stressed mice with yolkin resulted in the complete recovery of IL-6 production over the control values.

Fig. 4.

The ex vivo effects of oral yolkin administration during restraint stress and development of contact sensitivity on Con A-induced IL-6 production by splenocyte cultures. The treatment of mice was described in the legend to Fig. 2. The pooled splenocytes from four mice were isolated 24 h after elicitation of the CS. The results are presented as a mean value from triplicate measurements in pg/mL $\pm$ SD. * statistically significant (p 0.05).

As shown in Fig. 5A, at the time of CS induction, the levels of proliferation in Con A-stimulated splenocyte cultures were comparable in control and yolkin-treated mice. A lower level of proliferation of splenocytes from stressed mice was found. However, this effect was reversed in the stressed mice drinking the yolkin solution. In the case of LPS-induced splenocyte proliferation, there was no reduction in proliferation (Fig. 5B).

Fig. 5.

The ex vivo effects of oral yolkin administration during restraint stress and development of contact sensitivity on Con A-induced (A) and LPS-induced (B) splenocyte proliferation. The treatment of mice was described in the legend to Fig. 2. The pooled splenocytes from four mice (repeated 2 times) were isolated at 24 h after elicitation of the CS. The percentage of proliferation was calculated relative to the proliferation determined in the background group of mice. The results are presented as mean values of ten measurements $\pm$ SD. *** statistically significant (p 0.001).

The serum corticosterone levels in mice upon elicitation of the CS reaction are shown in Fig. 6. Its concentration was significantly elevated in the mice treated with yolkin alone but not in the stressed mice. However, the corticosterone levels were lower in the mice subjected to stress and treated with yolkin.

Fig. 6.

Serum corticosterone levels in stressed mice with developed contact sensitivity. The treatment of mice was described in the legend to Fig. 2. The peripheral blood was isolated from mice 24 h after elicitation of the CS. The results are presented as mean values in ng/mL $\pm$ SD. * and ** statistically significant (p 0.05 and p 0.01, respectively).

As shown in Fig. 7 the number of thymocytes, calculated as a ratio of cell number per body weight, in mice subjected to the immobilization stress, was decreased. The administration of yolkin to the unstressed mice did not change the thymocyte number. However, the treatment of stressed mice with yolkin caused a significant recovery of the thymocyte numbers.

Fig. 7.

The effects of immobilization stress on the number of thymocytes. The treatment of mice was described in the legend to Fig. 2. The thymuses were isolated from mice 24 h after elicitation of the CS. The results are presented as mean values of the thymocyte number index (thymocyte number $\times$10⁶/weight of mouse) $\pm$ SD. * statistically significant (p 0.05).

Figs. 8,9 illustrate the histological picture of auricles. Background (BG) group of mice represented mice sensitized to OXA, but instead receiving the eliciting dose of antigen they were treated only with acetone (vehiculum) onto the auricles. Therefore, they represent a group with nonspecific skin irritation. In the auricles from the BG group of mice (Fig. 8A and Fig. 9A), focal and weak inflammatory infiltrates, mostly composed of Gr-1 positive cells (neutrophils), were found. The infiltrates were present mainly near the base of the auricles. The inflammatory process occurred in the perivascular zone of the dermis and in the epidermis in the form of pustules, which were also Gr-1 positive in the immunohistochemical analysis. The morphometrical analysis revealed that pustules were in the early stage of formation, with the number of pustules and the area of lesions being the lowest in comparison to other groups (Table 1). Only very small subcorneal pustules were observed. Immunohistochemical staining revealed that the number of T lymphocytes was the smallest, but the number of mast cells was the highest of all the studied groups. The number of Langerhans cells counted in the high power field was comparable to other tested groups. Because of lack of significant differences in the contents of lymphocytes, mast cells and Langerhans cells between the studied groups, the results were not shown.

Fig. 8.

The histological examination of the auricles of the experimental mice. Mice were administered yolkin in their drinking tap water, at a concentration of 5 µg/mL, for 5 consecutive days when they were stressed. Then the mice were treated with OXA to develop the CS (as described in the legend to Fig. 2). The auricles were isolated from mice 24 h after elicitation of the CS. (A) BG group (weak, focal infiltrate). (B) Ctrl group (edema and extensive neutrophilic infiltration in the dermis). (C) Ctrl group (edema and infiltration in the dermis, medium-sized pustules in the epidermis). (D) Yolkin group (moderate infiltration in the dermis and small pustules in the epidermis). (E) Ctrl Stress group (severe infiltration and edema in the dermis, large pustules in the epidermis). (F) Yolkin/Stress group (moderate infiltration in the dermis, small and medium pustules in the epidermis). Arrows – pustules, arrowheads – infiltration, asterisks – edema; scale bar = 100 µm. BG, background group (mice given only the eliciting dose of the antigen).

Fig. 9.

The presence of neutrophils in the auricles of the experimental mice. Mice were administered yolkin in their drinking tap water, at a concentration of 5 µg/mL, for 5 consecutive days when they were stressed. Then the mice were treated with OXA to develop the CS (as described in the legend to Fig. 2). The auricles were isolated from mice 24 h after elicitation of the CS. Neutrophils were detected with rat anti-mouse Ly6G antibody (component of antigen Gr-1, clone RB6-8C5). (A) BG group, receiving only the eliciting dose of the antigen (very small subcorneal pustules). (B) Ctrl group, higher magnification of mouse auricle (strong infiltration in the subepidermal region of the dermis, and pustules in the epidermis). (C) Ctrl group (visible infiltration and medium sized and large pustules). (D) Yolkin group (moderate infiltration in the dermis and small pustules). (E) Ctrl Stress group (extensive infiltrate in the dermis and numerous large pustules). (F) Yolkin/Stress group (moderate infiltration in the dermis and small pustules in the epidermis); scale bar = 100 µm. BG, background group (mice given only the eliciting dose of the antigen).

In the control, immunized and non manipulated group (Fig. 8B,C and Fig. 9B,C), strong inflammatory infiltrates in the dermis and very numerous pustules (small, medium and large) in the epidermis, along the entire auricles, were registered (Table 1). Because of the intensification of the inflammatory process, the number of neutrophils increased twofold in comparison to the background group. The number of lymphocytes increased, compared to the background group, but the number of Langerhans cells and mast cells did not change. The morphometrical analysis revealed an increase in the thickness of auricles in the control group. On the H&E stained sections, a loosening of collagen fibers, indicating edema, was observed.

In the group of stressed mice, the inflammatory process in the dermis was comparable to the control group, with extensive, neutrophilic infiltrations in the dermis (Fig. 8E and Fig. 9E). The inflammatory process in the epidermis of stressed mice was more profound than in the unstressed group regarding area and number of pustules (Table 1). The auricle thickness was still significant and swelling of the connective tissue was visible, indicating edema. The numbers of T lymphocytes (CD3+), Langerhans (CD207+) and mast cells, were similar to those in the control group (data not shown).

In the group of mice treated with yolkin and yolkin-treated stressed mice, a diminution of the inflammatory process was observed (Fig. 8D,F and Fig. 9D,F). There were still moderate neutrophilic infiltrates in the auricle dermis, but was expressed less than in the control group and mice subjected to the stress. The number of neutrophils also decreased. The formation of neutrophilic pustules was limited to small and medium lesions (Table 1). In both groups the morphometric measurements demonstrated a decrease in the auricle thickness which indicates a reduction of edema. The number of lymphocytes, mast cells and Langerhans cells did not change in comparison to other groups.

The quantitative parameters of the inflamed auricles regarding the size and distribution of pustules is presented in Table 1.