Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.
Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day “0”, mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day “4”, the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into naïve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.
Allergic contact dermatitis (ACD) is a common skin inflammatory disease in industrialized countries caused by a type IV hypersensitivity reaction resulting from exposure to low molecular weight chemicals called haptens. The substances causing contact sensitization in humans include heavy metal ions (chromium, nickel, iron, cobalt), turpentine, fragrances, dyes, and preservatives present in cosmetics (e.g., p-phenylenediamine), some drugs (e.g., neomycin, benzocaine), β-lactam antibiotics (i.e., penicillin), chemicals produced by plants (pentadecacatechol, a substance present in poison ivy), as well as hydroquinone-used in the photographic industry1,2. ACD etiological agents are very high as over 100,000 chemicals are used in industry alone, and 2,000 new ones are synthesized each year. To date, more than 3,700 molecules have been identified that may be contact haptens/allergens3. The contact hypersensitivity reaction (CHS) is an experimental model of ACD that can be studied in mice, guinea pigs, and rats and can be induced by the topical skin application of reactive chemical haptens dissolved in organic solvents4,5,6. This study aims to describe an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests.
The CHS consists of sensitization (induction) and effector (challenge) phases. In animal models, haptens first bind covalently to proteins in the body to create neoantigens. During the sensitization phase, activated keratinocytes promote the migration and maturation of skin dendritic cells (sDCs) by producing pro-inflammatory cytokines-tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β)7. Epidermal Langerhans cells (LCs) present antigens during the CHS induction and effector phases8. LCs exposed to hapten during sensitization promote the induction of both regulatory and effector cells9. Increasing evidence from several studies suggests that CHS responses can be mediated by either CD4+ MHC class II-restricted Th1 cells, locally releasing interferon-γ (IFN-γ) to employ a characteristic inflammatory infiltrate, CD8+ MHC class I-restricted Tc1 lymphocytes that can also release IFN-γ but mostly mediate cytotoxic damage to keratinocytes, and now also interleukin 17 (IL-17)-producing Th17 cells10,11.
Several different CHS models employing various species12,13,14 and haptens have been developed (a detailed comparison of different haptens, solvents, and time of application is summarized in Table 1). The mouse, a frequently used laboratory species, offers a few advantages in studying CHS. There are more strains, knockouts (KO), and transgenic animals among mice compared to other species, which makes them a very attractive animal15. In addition, the CHS model requires many animals, and mice are more economical here. Animal models do not mimic ACD in all aspects; in particular, they show crusting and desquamation, which is not common for humans16,17. The features of chronic disease are challenging to reproduce, mainly because the described model does not assume the application of the hapten for a long period of time. However, it has been confirmed here that many of the significant aspects of ACD are reproduced. It has also been shown that, as in humans, these features are associated with local allergic reactions. The choice of hapten, its solvent, and its application outlined in this protocol were dictated by the fact that the results have been confirmed by numerous in vitro tests and that it was tested and modified in the laboratory for many years until the current version was established. Murine models allow for the analysis of the cell subsets or cytokines that are involved in the development of ACD and are essential for preclinical assessments of new treatments.
All experiments presented in this article were conducted according to the guidelines of the 1st Local Ethical Committee on Animal Testing in Krakow. All the procedures described were performed according to the local recommendations, especially regarding using ketamine/xylazine as an anesthetic, using both sides of the ears to apply the substance/hapten, cutting off the ear, and collecting blood by eyeball removal. BALB/c (haplotype H-2d), CBA/J (H-2k), and C57BL/6 (H-2b) male and female mice, 6-12 weeks old, were used for the present study (see Table of Materials). For statistical significance, it is best if each group of mice consists of 10-12 animals.
1. Animal preparation
2. Marking mice for identification
3. Induction of CHS
NOTE: This procedure is depicted in Figure 1.
4. Ear biopsies
5. Histology of ear tissue
6. Vascular permeability test
NOTE: Alternately to ear thickness measurement, a vascular permeability test can be performed.
7. Serum collection and anti-TNP immunoglobulin (IgG1) antibody measurement
8. Adoptive transfer of the CHS-effector cells
NOTE: This procedure is depicted in Figure 2.
For CHS induction, the animals were sensitized via skin painting (abdominal) with 150 µL of 5% TNCB or sham sensitized with the vehicle alone. On day "4", the ear swelling responses of both ears were induced by contact painting (challenge) with 10 µL of 0.4% TNCB in both mice previously contact sensitized with TNCB (test group) and the control group mice (sham sensitized). The presented data depict that the mice sensitized with TNCB and challenged 4 days later developed significantly increased ear swelling compared with the sham-sensitized ones similarly challenged (Figure 3, Table 2, test vs. control group). The ear swelling results were completely validated in further studies, highlighting that an increase in ear edema determined with a micrometer agreed with augmented ear weight (Figure 4), MPO activity (Figure 5), IFN-γ concentration in the ear extracts (Figure 6), increased thickening of the edematous dermis in the histological examination (Figure 7), and ear vascular permeability (Figure 8). An increase in the concentration of TNP-specific IgG1 antibodies was also found in the sera of the test mice when compared with the control animals (Figure 9).
As an example of T cell-mediated immune response, CHS can also be transferred into naïve syngeneic recipient mice. Donors were sensitized by TNCB application, and subsequently, the CHS-effector cells were administered i.v. into the naïve recipient mice, which were challenged with the hapten and tested for CHS 24 h later (Figure 10). The animals that received the CHS-effector cells from donors previously sensitized with TNCB showed significantly increased ear swelling compared to animals that were challenged only (did not receive any cells).
The CHS reaction has a complex mechanism and involves various cells. Antigen presentation and T/B cell activation occur in the peripheral lymph organs (e.g., ALNs and SPL). It was determined that CHS-effector cells depleted of CD4+ but not CD8+ cells prior to adoptive cell transfer resulted in the absence of the CHS reaction in the recipient mice. Those cells were found to be positive for IFN-γ (T-box transcription factor TBX21, Tbet+) and IL-17A (retinoic acid receptor-related orphan nuclear receptor gamma, RORγT+) (Supplementary Figure 1).
The presented results from the representative experiments were performed on C57BL/6 and CBA/J male and female mice at 8-12 weeks of age. Following the 3R rules in the use of animals23, especially reduction, for the purposes of this article, the results of experiments are shown on small groups of animals. Data in the graphs are shown as mean ± SEM. Statistical significance was set at p < 0.05. The graphs were drawn using Prism software (see Table of Materials).
Figure 1: Induction of CHS. Sensitization, challenge, and ear measurement. Abbreviations: CHS = contact hypersensitivity reaction; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 2: Adoptive transfer of the CHS-effector cells. Abbreviations: ALNs =axillary and inguinal lymph nodes; CHS = contact hypersensitivity reaction; i.v. = intravenously; SPLs = spleens; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 3: Representative evaluation of CHS to TNCB by the measurement of ear swelling with a micrometer. Mice were TNCB (test group) or sham (control group) sensitized and subsequently challenged. The thickness of the auricle was measured before and after the challenge, and differences in ear swelling were calculated by subtracting the 0 h ear thickness (µm) from the 24 h ear thickness (µm). Ear swelling was expressed as mean ± SEM, ****p < 0.0001, n = 10 mice/group (data from Table 2). Abbreviations: CHS = contact hypersensitivity reaction; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 4: Representative evaluation of CHS by the measurement of ear weight. Ear weight is one of the parameters that corresponds with ear swelling. Mice were TNCB (test group) or sham (control group) sensitized and subsequently challenged. At 24 h after the challenge, 6 mm diameter punches were taken from the removed ears. The punches were weighed on an analytical laboratory balance. Ear weight was expressed in milligrams (mg) as mean ± SEM, ***p < 0.001, n = 10 mice/group. Abbreviations: CHS = contact hypersensitivity reaction; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 5: Representative evaluation of MPO activity. Increased MPO activity in tissue extracts correlates with ear inflammation. TNCB-sensitized mice (test group) and sham-sensitized mice (control group) were challenged. At 24 h post-challenge, the ears were removed, and 6 mm diameter punches of the ear were extracted and processed. MPO activity is expressed in U per protein content (U/g of protein). Results displayed as mean ± SEM, **p < 0.01, n = 5-6 mice/ group. Abbreviations: CHS = contact hypersensitivity reaction; MPO = myeloperoxidase; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene; U = units. Please click here to view a larger version of this figure.
Figure 6: Representative evaluation of cytokine production-IFN-γ concentration in ear extracts. Mice were TNCB (test group) or sham (control group) sensitized and subsequently challenged. At 24 h post-challenge, the ears were removed, and 6 mm diameter punches of the ear were taken. The concentration of IFN-γ was determined in tissue homogenates by ELISA. Results shown as mean ± SEM, *p < 0.05, n = 5 mice/group. Abbreviations: CHS = contact hypersensitivity reaction; IFN-γ = interferon gamma; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 7: Representative histology of the ear tissue. Hematoxylin and eosin staining. Mice were TNCB (test group) or sham (control group) sensitized and subsequently challenged. (A–C) Histological examination in the test group manifested in a significantly increased concentration of inflammatory cells (mononuclear and polymorphonuclear cells), mainly in the dermis, with microabscess formation in the epidermis. Thickening of the edematous dermis and a thickened, hyperplastic epidermis were also noticed. (D–E) Control group. Please click here to view a larger version of this figure.
Figure 8: Representative vascular permeability test. Observed ear tissue edema is a result of increased vascular permeability. To determine changes in vascular permeability, mice were TNCB (test group) or sham (control group) sensitized and then challenged 4 days later. At 23 h after the challenge, Evans blue was injected, and, 1 h after Evans blue injection, the animals were euthanized, and 6 mm diameter punches of the ear were made. Results shown as mean ± SEM, **p < 0.01, n = 5 mice/group. Abbreviations: CHS = contact hypersensitivity reaction; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 9: Representative anti-TNP IgG1 antibody measurement. The concentration of anti-TNP IgG1 antibodies in serum was measured 24 h after the challenge with hapten TNCB in sham sensitized (control) and in TNCB-sensitized (test group) mice. The collected sera were tested for antibody concentration by ELISA. Results shown as mean ± SEM, ***p < 0.001, n = 10 mice/group. Abbreviations: CHS = contact hypersensitivity reaction; IgG1 = immunoglobulin G subclass 1; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Figure 10: Representative adoptive transfer of the CHS-effector cells. The CHS-effector cells were obtained from donors that were sensitized with TNCB. Next, the collected immune cells were injected, i.v., into naïve syngeneic recipients, which were challenged for elicitation of the CHS effector phase. The control group of mice did not receive any cells prior to the challenge. The thickness of the auricle was measured before and after the challenge. Results shown as mean ± SEM, ***p < 0.001, n = 7 mice/group. Abbreviations: CHS = contact hypersensitivity reaction; SEM = standard error of the mean; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to view a larger version of this figure.
Mouse strain | Sensitization solution (dose) on shaved abdomen |
Elicitation solution (dose) on both sides of ear/ s |
Sensitization / elicitation day | Refs | |||||||||||
BALB/c (H-2d); C57BL/6 (H-2b) TCRδ-/-, β2m-/-, CD1d-/- (B10 PL (H-2u background) |
25 μL of 0.5 % DNFB in acetone-olive oil mixture (ratio 4:1) | 5 μL of 0.1 % DNFB in acetone-olive oil mixture (ratio 4:1) | 0 / 5 | 22 | |||||||||||
C57BL/6 (H-2b) | 50 μL of 0.5 % DNFB in acetone-olive oil mixture (ratio 4:1) | 25 μL of 0.2 % DNFB in acetone-olive oil mixture (ratio 4:1) | 0 / 5 | 32 | |||||||||||
C57BL/6 (H2b); IL-17A-/- (C57BL/6 background) | 150 μL of 5% TNCB in acetone-ethanol mixture (ratio 1:3) | 10 μL of 0.4 % TNCB in olive oil-acetone mixture (ratio 1:1) | 0 / 4 | 33 | |||||||||||
CBA/J (H-2k); C57BL/6 (H-2b) TLR2-/-, MyD88-/-, IL-17A-/- (C57BL/6 background) |
150 μL of 5 % TNP-Cl (TNCB) in an acetone-ethanol mixture (ratio 1:3) | 10 μl of 0.4 % TNP-Cl (TNCB) in olive oil-acetone mixture (ratio 1:1) | 0 / 4 | 21 | |||||||||||
C57BL/6 (H-2b); BALB/c (H-2d) | 25 μL of 1 % TNCB in an acetone | 10 μL of 0.1 or 0.2 % TNCB in an acetone (and higher up to 1 %) | 0 / 7 | 34 | |||||||||||
C57BL/6 (H-2b); TLR2-/-/ TLR4-/- (double-knockout mice on C57BL/6 background) |
100 μL of 3 % TNCB in an acetone | 20 μL of 1 % TNCB in an acetone (just on the back side of ears) | 0 / 5 | 31 | |||||||||||
C57BL/6 (H-2b) MHC class II-deficient mice (C57BL/6 background) |
100 μL of 3 % TNCB in acetone-olive oil mixture (ratio 4:1) | 20 μL of 0.5 or 1 % TNCB in acetone-olive oil mixture (ratio 4:1) | 0 / 6 | 35 | |||||||||||
C57BL/6 (H-2b) | 100 μL of 7 % TNCB in an acetone | 20 μL 1 % TNCB in an acetone | 0 / 5 | 36 | |||||||||||
C57BL/6 (H-2b) | 100 μL 3 % OX in ethanol | 20 μL 1 % OX in an ethanol | 0 / 5 | ||||||||||||
C57BL/6 (H-2b); CD4-/-, CD8-/- (C57BL/6 background) | 25 μL of 0.5 % DNFB in acetone-olive oil (ratio 4:1) | 10 μL of 0.2 % DNFB in acetone-olive oil (ratio 4:1) | 0 / 5 | 10 | |||||||||||
C57BL/6 (H-2b); CD4-/-, CD8-/- (C57BL/6 background) | 150 μL of 3 % OX in alcohol-acetone (ratio 3:1) | 10 μL 1% OX in alcohol-acetone (ratio 3:1) | 0 / 5 | ||||||||||||
C57BL/6 (H-2b); C3H/HeN (H-2k); TLR4-/- (C3H/HeJ background); MyD88-/- (C57BL/6 background) | 100 mg/ ear of 10 % NiCl2 in white petrolatum on the dorsal side of both ears | 10 % NiCl2 in white petrolatum | 0,1,2 / 23, 24 | 37 | |||||||||||
NOD (H-2g7) MyD88-/- (NOD background) | 400 μL of 0.5 % FITC in acetone and dibutyl phthalate | 10 μL of 0.1 % FITC in acetone and dibutyl phthalate | 0 / 5 | 25 |
Table 1: Comparison of the CHS model in various studies. Abbreviations: DNFB = 1-fluoro-2,4-dinitrobenzene; FITC = fluorescein isothiocyanate; NiCl2 = nickel (II) chloride; TNCB = 2,4,6-trinitrochlorobenzene; TNP-Cl = trinitrophenyl chloride; OX = oxazolone. Please click here to download this Table.
Control group (negative) | Test group (CHS reaction) | ||||||
Mouse # ear: L, R | 0 h ear thickness [μm] | 24 h ear thickness [μm] | 24 h – 0 h ear thickness [μm] | Mouse # ear: L, R | 0 h ear thickness [μm] | 24 h ear thickness [μm] | 24 h – 0 h ear thickness [μm] |
1 L | 365 | 380 | 15 | 1 L | 345 | 427.5 | 82.5 |
1 R | 335 | 380 | 45 | 1 R | 340 | 455 | 115 |
2 L | 345 | 355 | 10 | 2 L | 355 | 475 | 120 |
2 R | 327.5 | 352.5 | 25 | 2 R | 342.5 | 457.5 | 115 |
3 L | 340 | 370 | 30 | 3 L | 340 | 460 | 120 |
3 R | 325 | 355 | 30 | 3 R | 345 | 495 | 150 |
4 L | 335 | 380 | 45 | 4 L | 357.5 | 432.5 | 75 |
4 R | 340 | 350 | 10 | 4 R | 335 | 402.5 | 67.5 |
5 L | 350 | 380 | 30 | 5 L | 335 | 387.5 | 52.5 |
5 R | 337.5 | 360 | 22.5 | 5 R | 335 | 425 | 90 |
6 L | 335 | 365 | 30 | 6 L | 350 | 430 | 80 |
6 R | 340 | 375 | 35 | 6 R | 342.5 | 405 | 62.5 |
7 L | 345 | 337.5 | 0 | 7 L | 340 | 502.5 | 162.5 |
7 R | 345 | 335 | 0 | 7 R | 327.5 | 447.5 | 120 |
8 L | 370 | 380 | 10 | 8 L | 327.5 | 515 | 187.5 |
8 R | 375 | 355 | 0 | 8 R | 327.5 | 540 | 212.5 |
9 L | 385 | 370 | 0 | 9 L | 330 | 415 | 85 |
9 R | 342.5 | 362.5 | 20 | 9 R | 327.5 | 390 | 62.5 |
10 L | 307.5 | 340 | 32.5 | 10 L | 337.5 | 445 | 107.5 |
10 R | 325 | 350 | 25 | 10 R | 352.5 | 455 | 102.5 |
Mean | 20.75 | Mean | 108.5 | ||||
± SEM | 3.245 | ± SEM | 9.565 |
Table 2: Representative example of calculating the difference in ear thickness in the effector phase of CHS. Calculating the difference in the thickness of the auricle before and after the challenge with the hapten: 24 h ear thickness (µm) – 0 h ear thickness (µm). Each ear counts as a separate measurement. Ear swelling expressed in micrometers (µm) ± SEM, n = 20. Abbreviations: L = left; R = right. Please click here to download this Table.
iSTD dilution with AD | anti-TNP IgG1 Ab (U/mL) |
100x | 250 |
200x | 125 |
400x | 62.5 |
800x | 31.25 |
1600x | 15.63 |
3200x | 7.8 |
only AD | 0 |
Table 3: Preparation of the different concentrations of iSTD for the standard curve for anti-TNP IgG1 Ab measurement. The 100x iSTD dilution was assumed to be 250 U of anti-TNP IgG1 Ab. Abbreviations: Ab = antibody; AD = assay diluent; iSTD = internal standard; IgG1 = immunoglobulin G subclass 1; TNP = 2,4,6-trinitrophenyl; U = units. Please click here to download this Table.
Supplementary Figure 1: The phenotype of CHS-effector cells. The CHS-effector cells were obtained from donors' ALNs and SPLs, which were previously sensitized with TNCB. (A) Employing the MACS technique, the CHS-effector cells (whole ALNs and SPLs) were depleted of either CD4+ or CD8+ cells. Subsequently, adoptive cell transfer was conducted prior to the elicitation of the CHS effector phase. Ear swelling was expressed as mean ± SEM. (B–E) Using a flow cytometry technique, the CHS-effector and naïve (obtained from naïve mice) cells were stained for IFN-γ, Tbet, IL-17A, and RORγt prior to analysis. Cells were gated for the TCRβ+CD4+population. Results shown as mean ± SEM, ***p < 0.001, **p < 0.01, * p < 0.05, n = 4-6 mice/group. Abbreviations: ALNs = axillary and inguinal lymph nodes; CD4 = cluster of differentiation 4; CHS = contact hypersensitivity reaction; IFN-γ = interferon gamma; IL = interleukin; MACS = magnetic-activated cell sorting; ns = non significant; RORγt = retinoic-acid-receptor-related orphan nuclear receptor gamma; SEM = standard error of the mean; SPLs = spleens; Tbet = T-box transcription factor TBX21; TCRβ = T cell receptor beta; TNCB = 2,4,6-trinitrochlorobenzene. Please click here to download this File.
CHS is induced via haptens, which bind to self-protein antigens in the skin, creating neoantigens. CHS is mediated by local extravascular recruitment of circulating antigen-specific CHS-effector T cells, which results in swelling in the challenged tissue, peaking 24 h after exposure of the secondary skin to the same hapten6. The swelling of the tissue is mainly caused by the infiltration of leukocytes and leukocyte-dependent fibrin deposition24. These changes can be detected with a micrometer measuring the ear swelling of hapten-sensitized and challenged versus sham-sensitized and challenged mice.
CHS can also be determined by the comparison of ear weight. Then, the ear punches utilized for ear weight determination can be used for further tests. The cell infiltrates in the inflamed ears consist of cytokine-producing T lymphocytes and MPO-positive neutrophils. In the ear tissue homogenates, various parameters can be measured, such as MPO activity or the concentration of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-17A, or others) using the ELISA test or the expression of the cytokine mRNA in the skin using the qPCR test25. Additionally, vessel permeability changes can be evaluated using the Evans blue test21,26.
In the inflamed ear tissue, the changes observed can also be complemented with in vitro tests, highlighting T cell proliferation and cytokine production. This can be readily accomplished by culturing ALNs in the presence of hapten-conjugated protein antigens22,27. The evaluation of cytokine secretion by ALNs isolated from sensitized but not challenged mice provides details regarding cytokine production by the CHS-effector cells in the place of their induction.
Limitations
Many micrometers measure ear swelling with differing accuracy. For example, the lowest pressure is exerted by a spring caliper, so it seems that the results will best reflect the actual thickness ear measurement. However, micrometers that exert more pressure, such as Mitutoyo, are likely to compress more tightly the fluid that accumulates in the ears in the early phase of edema formation. The biphasic nature of CHS can be visualized more clearly using such micrometers because more pressure is exerted. This is more difficult when using a spring caliper with light pressure28. Nevertheless, in some studies, the thickness of the ears was measured with a caliper29. Also, the observer's experience ensures accurate measurements, which can be influenced by subjective feelings, even if the observer is unaware of the experimental groups.
Alternative methods have been described here that can help confirm the measurements of ear swelling with a micrometer, making the data presented more reliable and less subjective. However, these alternative strategies for assessing CHS can only be used at one point in time. Micrometer measurements can be repeated at various time points, allowing for studying the CHS kinetics.
Modifications
The protocol for studying the CHS reaction we use in our laboratory differs significantly from those used in other laboratories, including the dose of hapten and the solvent composition used for both elicitation and sensitization, as well as the time point at which the reaction is assessed. The ear thickness can be measured at different time points (e.g., 2 h, 24 h, 48 h, and 72 h after the challenge)10,30. Table 1 shows different experimental models, particularly the differences in the animal strain, hapten, and the sensitization/challenge times used in various studies10,21,22,26,31,32,33,34,35,36,37. The CHS protocol might also be conducted without previous shaving of the abdomen of the mice.
The next difference concerns the execution of the elicitation (challenge) itself. As is typical, the sensitization is done by painting shaved mice's abdomen skin with the hapten or vehicle alone. Subsequently, in both groups, one ear is challenged with diluted hapten on each ear side. As a control, the opposite ear is painted with an identical amount of vehicle alone10,31.
Critical steps
The most critical moment is to trigger CHS, as the test results depend on it. (1) Hapten solution is very volatile and light sensitive, so it must be tightly closed and protected from light, and when in use, it must be quickly applied to the skin of the animals so it will not evaporate. (2) After applying the hapten to the skin of the abdomen, it must be dried before the animal returns to the cage because mice can smear it against the bedding or apply it to the ears with their paws (then the measurement of the thickness of the auricle may be inadequate). (3) Various methods of labeling animals, such as marking mouse tails with a solvent-resistant marker, are also known. However, the dyes present in the marker might (not studied) become a hapten and trigger CHS. Therefore, in this study, a marking method was chosen that does not affect the reaction. (4) Choosing a shaving method is very important as it can irritate the skin. In this protocol, a gray soap was used because it has soothing properties; it accelerates the treatment of minor cuts and festering wounds, which is helped by its antibacterial effects. It soothes swelling and inflammation of the skin.
Further investigations and standardization of the experimental procedures, including the animal use (strain and sex), are required to compare the results from different studies.
Many different environmental factors and new substances with biological functions can be tested in the CHS model. This model can be useful if researchers want to show if tested factors modulate T cell-dependent immune responses.
The authors have nothing to disclose.
This study was supported by subvention SUBZ.A020.22.060 of the Medical University in Wroclaw, Poland, and by grants from the Ministry of Science and Higher Education N N401 545940 to MS and IP2012 0443 72 to MMS.
70% ethanol | Merck KGaA, Darmstadt, Germany | 65350-M | for surface disinfection |
96-well flat-bottom plates, polypropylene | Greiner Bio-One GmbH, Kremsmunster, Austria | 655101 | for MPO and Evans blue measurement – plates should be made of polypropylene, that has a lower binding capacity so proteins or DNA will not bind |
Acetone (ACS reagent, ≥99.5%) | Merck KGaA, Darmstadt, Germany | 179124 | |
Aluminum foil | Merck KGaA, Darmstadt, Germany | Z185140 | |
Analytical balance | Sartorius Weighing Technology GmbH, Goettingen, Germany | PRACTUM224-1s, 29105177 | |
Automated tissue processor | MediMeas Instruments, Sarsehri, Haryana, India | MTP-E-212 | automatically prepare tissue samples by fixing, dehydrating, clearing, and infiltrating them with paraffin |
BD Vacutainer SST II Advance (tube with gel for obtaining serum) | Becton Dickinson (BD), Franklin Lakes, NJ, USA | BD 366882 | |
Bicinchoninic acid kit for protein determination | Merck KGaA, Darmstadt, Germany | BCA1-1KT | |
Biotin Rat Anti-Mouse IgG1 | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 553441 | |
BSA (bovine serum albumine) | Merck KGaA, Darmstadt, Germany | A9418 | protein assays & analysis, 2 mg/mL |
Cell strainer, pore size 70 μm | BIOLOGIX, China | 15-1070 | suitable for 50 mL tubes |
Coverslip | VWR, Radnor, Pennsylvania, United States | 631-1583 | 24 mm, but it possible to use different size |
Disposable pipettes capacity: 5 mL, 10 mL, 25 mL | Merck KGaA, Darmstadt, Germany | Z740301, Z740302, Z740303 | |
DPBS (Dulbecco′s phosphate buffered saline) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 14190144 | no calcium, no magnesium, mammalian cell culture |
DPX Mountant for histology | Merck KGaA, Darmstadt, Germany | 6522 | mounting media for H-E, might be used some other e.g, Canada balsam |
Dumont 5 tweezers – straight | Animalab, Poznan, Poland | 11251-10FST | surgical instruments for procedures on mice (should be steriled) |
Dumont 7 tweezers – bent | Animalab, Poznan, Poland | 11272-50FST | surgical instruments for procedures on mice (should be steriled) |
Eosin Y solution, alcoholic | Merck KGaA, Darmstadt, Germany | HT110116 | |
Eppendorf Safe-Lock Tubes 1.5 mL | Eppenforf, Germany | 3,01,20,086 | polypropylene |
Eppendorf Safe-Lock Tubes, 2.0 mL | Eppenforf, Germany | 3,01,20,094 | polypropylene, round bottom (the homogenization beads can easily move) |
Ethanol 100% (absolute alkohol) | Merck KGaA, Darmstadt, Germany | 1.07017 | |
Ethanol 96% | Merck KGaA, Darmstadt, Germany | 1.59010 | |
Evans blue | Merck KGaA, Darmstadt, Germany | E2129 | |
FBS (fetal bovine serum) | ThermoFisher Scientific, Waltham, Massachusetts, USA | A3160802 | |
Formalin solution, neutral buffered, 10% | Merck KGaA, Darmstadt, Germany | HT501128 | |
Formamide 99.5% (GC) | Merck KGaA, Darmstadt, Germany | F7503 | |
Freezer -20 °C | Bosch, Germany | GSN54AW30 | |
Fridge +4 °C / freezer -20 °C | Bosch, Germany | KGV36V10 | mammalian Cell Culture, qualified, Brazil, 10 x 50 mL |
Glass microskope slides, SuperFrost Plus | VWR, Radnor, Pennsylvania, United States | 631-0108, 631-0446, 631-0447, 631-0448, 631-0449 | Slides that eliminates the need to apply adhesive materials or protein coatings, to preventing any tissue sections loss during staining. |
Graph Pad Prism | GraphPad Software Inc. | v. 9.4.0 | |
Grey soap | Pollena Ostrzeszów, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland | Bialy jelen soap bar | grey Soap Bar Natural Hypoallergenic. Generally available product |
H2SO4 (sulfuric acid) 1 mol/l (1 M) | Merck KGaA, Darmstadt, Germany | 1.60313 | |
Harris hematoxylin solution | Merck KGaA, Darmstadt, Germany | HHS16 | |
Hemocytometer | VWR, Avantor, U.S.A | 612-5719 | manual counting chamber is recommend, which is more accurate |
Hexadecyltrimethylammonium bromide | Merck KGaA, Darmstadt, Germany | H5882 | |
Homogenizer | QIAGEN Hilden, Germany | Tissue Lyser LT, SN 23.1001/05234 | homogenizer with stainless steel beads (diameter 5 mm) for 2 mL centrifuge tubes |
Horseradish peroxidase streptavidin (HRP streptavidin) | Vector Laboratories, Inc., Burlingame, CA, USA | SA-5004-1 | |
Hydrogen peroxide solution (H202) | Merck KGaA, Darmstadt, Germany | H1009 | |
Incubator Heracell 150i | Thermo Electron LED Gmbh, Germany | 41071068 | 37 oC in the atmosphere of 5 % CO2, and 65 0C for deparaffinization the sections for histology |
Ketamine 100 mg/mL, solution for injection | Biowet Pulawy Sp. z o.o., Pulawy, Poland | cat.# not avaliable | |
KH2PO4 (potassium dihydrogen phosphate) 99.995% anhydrous basis | Merck KGaA, Darmstadt, Germany | 1.05108 | |
Laboratory Centrifuge | Heraeus Megafuge 1.0R, Thermo Scientific, Germany | B00013899 | speed to 300 x g, with cooling to 4 0C |
Laboratory Centrifuge | Heraeus Fresco 21, Thermo Scientific, Germany | 75002425 | speed to 3,000 x g, with cooling to 4 0C |
Mask (FFP2) | VWR, Radnor, Pennsylvania, United States | 111-0917 | for working with ortho-dianisine dihydrochloride |
Mice | Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland | CBA/J, C57BL/6 | |
Micrometer | Mitutoyo, Tokyo, Japan | 193-111 | digit Outside Micrometer, Ratchet Stop, 0-25mm Range, 0.001mm Graduation, +/-0.002mm Accuracy, https://shop.mitutoyo.pl/web/mitutoyo/pl_PL/all/all/Mikrometr%20analogowy%20/PR/193-111/index.xhtml |
microplate, 96 well, microlon, high binding (for ELISA test) | Greiner Bio-One GmbH, Kremsmunster, Austria | 655061 | with maxi-sorp binding surfaces for reliable and reproducible results in colormetric assays |
Microscope with objectives | Leica Microsystems CMS GmbH, Germany | DM1000, 294011-082007 | histology presented in the paper was performed under ThermoFisher Scientific EVOS M5000 Imaging System, with objectives: FL 20X LWDPH, 0.40NA/3.1WD and FL 40X LWDPH 0.65NA/1.79WD |
Myeloperoxidase from human leukocytes (MPO standard) | Merck KGaA, Darmstadt, Germany | M6908 | |
Na2HPO4 x 7 H2O (sodium phosphate dibasic heptahydrate) | Merck KGaA, Darmstadt, Germany | S9390 | |
Olive-oil | Merck KGaA, Darmstadt, Germany | 75343 | pure, natural |
OptEIA Mouse IFN-γ ELISA Set | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 555138 | |
Ortho-dianisine dihydrochloride | Merck KGaA, Darmstadt, Germany | D3252 | |
Paraffin wax | Merck KGaA, Darmstadt, Germany | 76242 | beads, waxy solid |
PBS (phosphate buffered saline) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 20012027 | pH 7.2, mammalian cell culture |
ph meter | Elmetron, Poland | CP-401 | |
Pipettes, variable volume with tips | Merck KGaA, Darmstadt, Germany | EP3123000900-1EA | 3-pack, Option 1, 0.5-10 uL/10-100 uL/100-1000 uL, includes epT.I.P.S. |
Razor blade | VWR, Radnor, Pennsylvania, United States | PERS94-0462 | scraper and cutter blades, single edge, aluminium spine, 100 blades per box, individually wrapped |
Rotary microtome | MRC Laboratory-Instruments, Essex, CM20 2HU UK | HIS-202A | |
Scissors – straight, sharp / sharp | Animalab, Poznan, Poland | 14060-10FST | Surgical instruments for procedures on mice (should be steriled) |
Screw cap (open top) | Merck KGaA, Darmstadt, Germany | 27056 | black polypropylene hole cap, for use with 22 mL vial with 20-400 thread |
Spectrophotometer | BioTek Instruments, U.S.A | 201446 | universal microplate spectrophotometer: λ range: 200 – 999 nm, absorbance measurement range: 0.000 – 4.000 Abs |
Staining dish 20 slides with rack | Merck KGaA, Darmstadt, Germany | S6141 | e.g. 20 slide staining dishes complete with covers, slide rack and handle |
Sterile Disposable Biopsy Punch 6mm | Integra LifeSciences, Princeton, NJ, USA | 33-36 | |
Surgical scissors | Animalab, Poznan, Poland | 52138-46 | surgical instruments for procedures on mice (should be steriled) |
Tissue processing cassettes | Merck KGaA, Darmstadt, Germany | Z672122 | tissue processing/ embedding cassettes with lid |
TMB Substrate Reagent Set | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 555214 | |
TNCB (2,4,6-trinitrochlorobenzene) | Tokyo Chemical Industry CO., LTD, Japan | C0307 | |
TNP-BSA (bovine serum albumin conjugated with 2,4,6-trinitrophenyl) | Biosearch Technologies LGC, Petaluma, CA, USA | T-5050 | |
T-PER (tissue protein extration reagent) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 78510 | |
Tubes 15 mL sterile | Merck KGaA, Darmstadt, Germany | CLS430055 (Corning) | polypropylene, conical bottom |
Tubes 50 mL, sterile | Merck KGaA, Darmstadt, Germany | CLS430290 (Corning) | polypropylene, conical bottom |
Tween 20 | Merck KGaA, Darmstadt, Germany | P1379 | |
Vials, screw top, clear glass (vial only) 22 mL | Merck KGaA, Darmstadt, Germany | 27173 | for the preparation of hapten, screwed on so that it does not evaporate |
Water bath | AJL Electronic, Poland | LW102 | |
Wax (paraffin) dispenser | VWR, Radnor, Pennsylvania, United States | 114-8737 | |
Xylazine (xylapan 20 mg/mL) solution for injection | Vetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Poland | cat.# not avaliable | |
Xylene (histological grade) | Merck KGaA, Darmstadt, Germany | 534056 |