Topical antihistamines display potent anti-inflammatory activity linked in part to enhanced permeability barrier function

Abstract

Systemic antagonists of the histamine type 1 and 2 receptors (H1/2r) are widely used as anti-pruritics and central sedatives, but demonstrate only modest anti-inflammatory activity. Because many inflammatory dermatoses result from defects in cutaneous barrier function, and because keratinocytes express both Hr1 and Hr2, we hypothesized that H1/2r antagonists might be more effective if they were used topically to treat inflammatory dermatoses. Topical H1/2r antagonists additively enhanced permeability barrier homeostasis in normal mouse skin by the following mechanisms: (i) stimulation of epidermal differentiation, leading to thickened cornified envelopes; and (ii) enhanced epidermal lipid synthesis and secretion. As barrier homeostasis was enhanced to a comparable extent in mast cell-deficient mice, with no further improvement following application of topical H1/2r antagonists, H1/2r antagonists likely oppose mast cell-derived histamines. In four immunologically diverse, murine disease models, characterized by either inflammation alone (acute irritant contact dermatitis, acute allergic contact dermatitis) or by prominent barrier abnormalities (subacute allergic contact dermatitis, atopic dermatitis), topical H1/2r agonists aggravated, whereas H1/2r antagonists improved, inflammation and/or barrier function. The apparent ability of topical H1r/2r antagonists to target epidermal H1/2r could translate into increased efficacy in the treatment of inflammatory dermatoses, likely due to decreased inflammation and enhanced barrier function. These results could shift current paradigms of antihistamine utilization from a predominantly systemic to a topical approach.

Conflict of interest statement

The authors state no conflict of interest.

Figures

Figure 1. Topical H1/2r Antagonists Enhance Permeability Barrier Homeostasis in Normal Skin

The flanks of hairless mice (n=4–5 each) were treated with topical applications of either diphenhydramine chlorhydrate (H1r), cimetidine (H2r), or ethanol (vehicle) twice-daily for four days (see Methods for further details). At the end of treatments, changes in basal barrier function, assessed as transepidermal water loss (TEWL, a), surface pH (b), and electrical conductance (SC hydration, c) were measured. (d) Barrier disruption was induced by sequential cellophane tape strippings until TEWL levels ≥ 10-fold increase over baseline, and barrier recovery rates were assessed two and four hrs later (a=p<0.05 vs. vehicle, b=p<0.01 vs. vehicle, c=p<0.001 vs. vehicle, and d=p<0.001 vs. H1r+H2r)66. e: Percent barrier recovery was compared in mast cell-deficient KitW/KitW-v double heterozygote mice (MCDM) vs. wild-type (WBB6F1) mice two and four hrs after tape stripping, as above.

Figure 2. Topical H1/H2r Antagonists Stimulate Epidermal Differentiation

Hairless mice were treated as above, and paraffin-embedded sections (6 μm) then were immunostained to detect changes in filaggrin, involucrin, and loricrin content and localization (a-i). j: In parallel, mRNA was isolated from freshly-obtained epidermal sheets after treatments as in Fig. 1, above (n=4), and changes in mRNA levels for filaggrin, loricrin, and involucrin were assessed by rt(Q)-PCR (see Methods and suppl. Table 2 for further details). Scale bar = 50 μm.

Figure 3. Topical H1r/H2r Antagonists Enhance Corneocyte Envelope Thickness

Electron micrographs of biopsies of H1/2r antagonist-treated skin were processed for electron microscopy (a-c), as in Methods. d: 10 micrographs each of perpendicular sections taken at random from 6 different biopsy samples. CE dimensions were measured directly on the electron microscope, as in Methods. a-c, Osmium tetroxide post-fixation; Scale bar = 100μm.

Figure 3. Topical H1r/H2r Antagonists Enhance Corneocyte Envelope Thickness

Electron micrographs of biopsies of H1/2r antagonist-treated skin were processed for electron microscopy (a-c), as in Methods. d: 10 micrographs each of perpendicular sections taken at random from 6 different biopsy samples. CE dimensions were measured directly on the electron microscope, as in Methods. a-c, Osmium tetroxide post-fixation; Scale bar = 100μm.

Figure 4. Global Stimulation of Epidermal Lipid Synthesis by Topical H1/2r Antagonists

Hairless mice were treated as above (Fig. 1), and biopsies were snap-frozen in liquid nitrogen. Frozen sections (5 μm) were incubated with either the fluorophore, nile red, or vehicle, and viewed in a fluorescence microscope, as in Methods (a-c; arrows depict sites of enhanced staining for lipids in epidermis). d: Freshly-obtained, full-thickness skin biopsies were incubated with 14C-acetate, followed by epidermal isolation, lipid extraction, saponification, fractionation by thin layer chromatography, and quantitation of changes in non-saponifiable (NSL) and saponifiable (total fatty acid) lipids, as in Methods. e-j: mRNA was isolated from epidermal sheets as above (n=4 mice each), and changes in mRNA levels for lipid synthetic enzymes (HMGCoA reductases, serine palmitoyl transferase [SPT], fatty acid modifying enzyme, fatty acid 2-hydroxylase [FA2H], and the acylceramide-generating elongation of very-long chain fatty acid-4 [ELOVL4]) were assessed by rt(Q)-PCR, as in Methods and suppl. Table 2. Scale bar = 10μm.

Figure 5. Topical H1/2r Antagonists Stimulate Lamellar Production and/or Secretion

Biopsies of H1/2r antagonist- and vehicle-treated skin samples (as in Fig. 1) were processed for electron microscopy, as in Methods. Solid arrows point to individual or aggregated lamellar bodies, and open arrows depict changes in secreted contents at stratum granulosum (SG)-SC interface. Representative samples of outer epidermis of vehicle-treated (Veh)- (a), H1r antagonist (diphenhydramine chlorhydrate)-treated- (b+c), and H2r (cimetidine)-treated- (d) skin. Note increased organelle density in the SG of H2r-treated epidermis, and premature secretion of lamellar bodies in H1r antagonist-treated skin, (b-e), as well as enhanced secretion of lamellar body contents at the stratum granulosum (SG)-SC interface (H2r>H1r). f: Enhanced formation of lamellar bodies correlated with increased mRNA levels of ABCA12 in H2r antagonist-treated skin (n=4); the increase in ABCA12 mRNA following H1r antagonist treatments did not achieve statistical significance. a-e: Osmium tetroxide post-fixation. Scale bar = 0.5 μm.

Figure 5. Topical H1/2r Antagonists Stimulate Lamellar Production and/or Secretion

Biopsies of H1/2r antagonist- and vehicle-treated skin samples (as in Fig. 1) were processed for electron microscopy, as in Methods. Solid arrows point to individual or aggregated lamellar bodies, and open arrows depict changes in secreted contents at stratum granulosum (SG)-SC interface. Representative samples of outer epidermis of vehicle-treated (Veh)- (a), H1r antagonist (diphenhydramine chlorhydrate)-treated- (b+c), and H2r (cimetidine)-treated- (d) skin. Note increased organelle density in the SG of H2r-treated epidermis, and premature secretion of lamellar bodies in H1r antagonist-treated skin, (b-e), as well as enhanced secretion of lamellar body contents at the stratum granulosum (SG)-SC interface (H2r>H1r). f: Enhanced formation of lamellar bodies correlated with increased mRNA levels of ABCA12 in H2r antagonist-treated skin (n=4); the increase in ABCA12 mRNA following H1r antagonist treatments did not achieve statistical significance. a-e: Osmium tetroxide post-fixation. Scale bar = 0.5 μm.

Figure 6. Topical H1/2r Antagonists Improve Inflammatory Dermatosis, Independent of Benefits for Barrier Function

a: Changes in ear thickness in acute irritant and acute allergic contact dermatitis (AICD and AACD, respectively) 16 hrs after prior topical application of the H1/2r antagonists. b: Changes in transepidermal water loss 16 hrs after a single topical application of the H1r or H2r antagonist or vehicle to opposing flanks of previously-sensitized and subsequently challenged mice (3× = subacute allergic contact dermatitis [SACD]); (atopic dermatitis [AD] =10 hapten challenges). Dotted line indicates upper level of water loss in normal mice.