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. 2010 Nov;177(5):2563-75.
doi: 10.2353/ajpath.2010.090941. Epub 2010 Oct 1.

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo

Stephan Seeliger  1 , Jörg Buddenkotte, Anjona Schmidt-Choudhury, Carine Rosignoli, Victoria Shpacovitch, Ulrike von Arnim, Dieter Metze, Roman Rukwied, Martin Schmelz, Ralf Paus, Johannes J Voegel, Wolfgang E Schmidt, Martin Steinhoff
Affiliations

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo

Stephan Seeliger et al. Am J Pathol. 2010 Nov.

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

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Figures

Figure 1
Figure 1
Changes in intrarectal body temperature [ΔT (°C)] compared with basal value (0 = 33.51 ± 0.25°C). A: Continuous infusion of 100 pmol/kg b.w./h PACAP1-27. Mean ± SEM, *P < 0.05 (n = 10). B: Continuous infusion of 7.5, 15, or 30 pmol/kg b.w./h PACAP1-27. Mean ± SEM, *P < 0.05 (n = 9). Mean time until first flush symptoms occurred was 46.03 ± 5.30 minutes. C: Bolus injection of 315 pmol/kg b.w. PACAP1-27. Mean ± SEM, *P < 0.05 (n = 8). D: Different conditions and concentrations of continuous infusion. Mean ± SEM, *P < 0.05 (n = 10). E: Changes in intrarectal body temperature [ΔT (°C)] compared to basal (32.75 ± 0.25°C) after continuous infusion of 20 or 100 pmol/kg b.w./h VIP1-28. Mean ± SEM, *P < 0.05 (n = 7). F: Normal skin in a healthy volunteer before intravenous application of PACAP1-27. G: Flush phenomenon in a healthy volunteer after intraveneous application of PACAP1-27. Cutaneous erythema and facial as well as a marked periorbital edema was observed within minutes, with a peak at 30 minutes after application (representative from n = 33 volunteers).
Figure 2
Figure 2
A: Sequence of laser Doppler images recorded in response to intradermal injections of saline (upper panel) or PACAP at various doses (10−9–10−6 mol/L; panels 2–5). Laser Doppler images were obtained at 30-second intervals for 5 minutes. Flare development is indicated by color code where dark blue represents baseline. PACAP concentration-dependently mediates very strong vasodilatation (panels 3–5, concentration as indicated). White line represents 1-cm scale (data not normalized). Displayed series of laser Doppler images represents results of six independent studies. B: Concentration-dependent flare response in human skin to intradermal injection of PACAP (n = 6).
Figure 3
Figure 3
Double-immunofluorescence staining for PACAP (polyclonal, green) and mast cell tryptase (monoclonal, red) in tissues from patients with urticaria (n = 6). A: Marked immunostaining for PACAP (green) in nerve fibers close to the dermal-epidermal border of human skin tissue (arrows). In the environment of epidermal nerve fibers, single PACAP-positive dendritic-like cells (Langerhans cells) of urticaria patients were detected (arrowhead, red; ×20; Scale bar = 40 μm). B: In the upper dermis, marked immunofluorescence staining for PACAP in nerve fibers (arrows) closely accompanied by tryptase-positive mast cells (red; ×40; Scale bar = 31.5 μm). C: PACAP-positive nerve fibers (arrows) close to dermal blood vessels (arrowhead, ×40; Scale bar = 31.5 μm). D: Negative control (pre-immune absorption control) showing absence of PACAP in nerve fibers and endothelial cells demonstrating specificity of immunostaining (×10; Scale bar = 45 μm).
Figure 4
Figure 4
Immunohistochemical detection of VPAC1R in urticaria human skin (n = 6). A: Overview shows intense dermal staining of VPAC1R in blood vessels and occasionally in leukocytes and weak staining for VPAC1R in keratinocytes and certain fibroblasts (×10; Scale bar = 45 μm). B: Higher magnification shows intense staining of endothelial cells, mast cells and lymphocytes for VPAC1R (arrows) (×20; Scale bar = 40 μm). C: Weaker staining for VPAC1R in the deeper dermis, small blood vessels, and leukocytes (arrows) as compared to superficial dermis (×40; Scale bar = 31.5 μm). D and E: Negative to weak staining for VPAC1R in dermal connective tissue, and occasionally staining of lymphocytes and mast cells (arrows, ×40; Scale bar = 31.5 μm). F: Control tissue (pre-immune absorption) demonstrates absence of VPAC1R in the skin, verifying specificity (×10; Scale bar = 45 μm). Experiments were performed as described in Materials and Methods.
Figure 5
Figure 5
Immunohistochemical distribution of VPAC1R in the tissue of patients with atopic dermatitis (n = 6). A: Intensive staining of endothelial cells, keratinocytes and leukocytes was observed (arrows, ×20; Scale bar = 40 μm). B: Higher magnification shows intensive staining for VPAC1R in dermal endothelial cells (arrows, ×40; Scale bar = 31.5 μm). C: Control tissue (pre-immune absorption) shows negative staining for VPAC1R (×10; Scale bar = 45 μm). In addition, no immunoreactivity was observed in human skin endothelium using VPAC2R or PAC1R antibodies (data not shown).
Figure 6
Figure 6
Double immunofluorescence of VPAC1R (green) in human skin sections of patients with atopic dermatitis (n = 6). A: Colocalization of VPAC1R and CD31+ in dermal vascular endothelial cells of atopic dermatitis patients (arrow). No staining of smooth muscle cells against VPAC1R was seen. B: Membrane staining for VPAC1R (green) in endothelial cells of the same postcapillary venule as Figure 6A (arrow). C: Colocalization of VPAC1R (green) in many, but not all, CD4+ T-helper cells (yellow, arrow) of dermal T-cell infiltrate (stained against CD4+, red). D: Immunofluorescence of VPAC1R (green) in the same infiltrate as (A), but now against CD8+ T-cells. Rare colocalization (yellow, arrow) of VPAC1R (green) in CD8+ cytotoxic T cells (red) of the infiltrate E: No colocalization of VPAC1R (green) in tissue macrophages within dermal infiltrate stained against CD68 (red, arrow). F: Weak colocalization (yellow) in a few mast cells stained for tryptase (red, arrow) and VPAC1R (green). G: Localization of VPAC1R in basal and suprabasal keratinocytes (green, arrow). H: Staining of endothelial cells (green) for VPAC1R (arrow). Note surrounding CD4+ T cells (red), colocalization in yellow. I: Control tissue (preimmune absorption) demonstrates absence of VPAC1R in the skin, verifying specificity. Experiments were performed as described in Materials and Methods. Magnification, ×100; Scale bars = 12 μm.
Figure 7
Figure 7
In vitro measurement of changes in cAMP concentration in HDMEC after stimulation with the agonist VIP1-28 or PACAP1-38. Results show the concentration-dependent activation of VPAC1R in HDMEC by VIP1-28 and PACAP1-38, in contrast to the VPAC1R antagonists VIP6-28 and PACAP6-27, because PAC1R and VPAC2R are not expressed by HDMEC. *The change in cAMP concentration in HDMEC was reduced by approximately 50% by the inhibitor PACAP6-27 (Materials and Methods). This proves the functionality of VPAC1R. **After the application of PACAP 10−9 mol/L, the change in cAMP concentration in HDMEC was more than fourfold compared with the negative control. Experiments were performed at least three times.
Figure 8
Figure 8
PACAP-stimulated organ-cultured skin show increased number of CD31+ vessel cross-sections. Three millimeter punch biopsies from the upper arm of healthy volunteer subjects (n = 3) were placed in organ culture for 24 hours and treated with either (A and C) vehicle or (B and C) 100 nmol/L PACAP. Thereafter, paraffin sections were subjected to immunohistochemical staining for CD31. (Student’s t-test, significant differences compared with vehicle control at ***P < 0.001).
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References

    1. Steinhoff M, Stander S, Seeliger S, Ansel JC, Schmelz M, Luger T. Modern aspects of cutaneous neurogenic inflammation. Arch Dermatol. 2003;139:1479–1488. - PubMed
    1. Cevikbas F, Steinhoff A, Homey B, Steinhoff M. Neuroimmune interactions in allergic skin diseases. Curr Opin Allergy Clin Immunol. 2007;7:365–373. - PubMed
    1. Blais C, Jr, Rouleau JL, Brown NJ, Lepage Y, Spence D, Munoz C, Friborg J, Geadah D, Gervais N, Adam A. Serum metabolism of bradykinin and des-Arg9-bradykinin in patients with angiotensin-converting enzyme inhibitor-associated angioedema. Immunopharmacology. 1999;43:293–302. - PubMed
    1. Nussberger J, Cugno M, Cicardi M, Agostoni A. Local bradykinin generation in hereditary angioedema. J Allergy Clin Immunol. 1999;104:1321–1322. - PubMed
    1. Doutre M. Physiopathology of urticaria. Eur J Dermatol. 1999;9:601–605. - PubMed

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