Substance P Is Upregulated in the Serum of Patients with Chronic Spontaneous Urticaria

angioedema activity score angioedema quality of life questionnaire chronic spontaneous urticaria chronic urticaria quality of life questionnaire Substance P urticaria activity score urticaria control test TO THE EDITOR Chronic spontaneous urticaria (CSU) is characterized by short-lived itchy wheals, angioedema, or both for more than 6 weeks. Symptoms of CSU are mainly mediated by the release of histamine from mast cells, but as of yet it is not entirely understood how mast cells get activated in CSU. In most CSU patients, underlying causes such as intolerance to foodstuff, chronic infections, or autoreactivity (ie due to circulating IgG anti-FcεRI, IgG anti-IgE, or IgE antibodies to autoantigens) can be identified (Grattan et al., 1986Grattan C.E. Wallington T.B. Warin R.P. et al.A serological mediator in chronic idiopathic urticaria—a clinical, immunological and histological evaluation.Br J Dermatol. 1986; 114: 583-590Crossref PubMed Scopus (261) Google Scholar; Gruber et al., 1988Gruber B.L. Baeza M.L. Marchese M.J. et al.Prevalence and functional role of anti-IgE autoantibodies in urticarial syndromes.J Invest Dermatol. 1988; 90: 213-217Abstract Full Text PDF PubMed Scopus (281) Google Scholar; Hide et al., 1993Hide M. Francis D.M. Grattan C.E. et al.Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria.N Engl J Med. 1993; 328: 1599-1604Crossref PubMed Scopus (815) Google Scholar; Wedi et al., 1998Wedi B. Wagner S. Werfel T. et al.Prevalence of Helicobacter pylori-associated gastritis in chronic urticaria.Int Arch Allergy Immunol. 1998; 116: 288-294Crossref PubMed Scopus (158) Google Scholar; Magerl et al., 2010Magerl M. Pisarevskaja D. Scheufele R. et al.Effects of a pseudoallergen-free diet on chronic spontaneous urticaria: a prospective trial.Allergy. 2010; 65: 78-83Crossref PubMed Scopus (105) Google Scholar; Altrichter et al., 2011Altrichter S. Peter H.J. Pisarevskaja D. et al.IgE mediated autoallergy against thyroid peroxidase—a novel pathomechanism of chronic spontaneous urticaria?.PloS One. 2011; 6: e14794Crossref PubMed Scopus (193) Google Scholar; Hatada et al., 2013Hatada Y. Kashiwakura J. Hayama K. et al.Significantly high levels of anti-dsDNA immunoglobulin E in sera and the ability of dsDNA to induce the degranulation of basophils from chronic urticaria patients.Int Arch Allergy Immunol. 2013; 161: 154-158Crossref PubMed Scopus (66) Google Scholar; Zuberbier et al., 2014Zuberbier T. Aberer W. Asero R. et al.The EAACI/GA2LEN/EDF/AAAAI/WAO Guideline for the definition, classification, diagnosis and management of Urticaria The 2013 revision and update.Allergy. 2014; 69: 868-887Crossref PubMed Scopus (852) Google Scholar). Why some people in whom these potential causes are found develop urticaria and others don't is unclear, as is the question why CSU shows spontaneous remission at some point. Disease activity of CSU is assessed using the UAS (urticaria activity score) and the AAS (angioedema activity score), and the impact of CSU on quality of life is assessed using the CU-Q2oL (chronic urticaria quality of life questionnaire) and the AE-QoL (angioedema quality of life questionnaire) (Zuberbier et al., 2014Zuberbier T. Aberer W. Asero R. et al.The EAACI/GA2LEN/EDF/AAAAI/WAO Guideline for the definition, classification, diagnosis and management of Urticaria The 2013 revision and update.Allergy. 2014; 69: 868-887Crossref PubMed Scopus (852) Google Scholar). The recently introduced urticaria control test (UCT) enables a quick assessment of treatment efficacy and disease control (Weller et al., 2014Weller K. Groffik A. Church M.K. et al.Development and validation of the urticaria control test: a patient-reported outcome instrument for assessing urticaria control.J Allergy Clin Immunol. 2014; 133: 1365-1372Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). All of these tools are, however, based on the subjective assessment of symptoms by the patient, and there is currently no objective measure to assess disease activity or severity. We and others have tried in the past to identify biomarkers in CSU. Among these, D-dimer has been shown to correlate with disease severity and to be especially high in antihistamine-resistant urticaria (Asero et al., 2008Asero R. Tedeschi A. Riboldi P. et al.Severe chronic urticaria is associated with elevated plasma levels of D-dimer.Allergy. 2008; 63: 176-180PubMed Google Scholar; Asero, 2013Asero R. D-dimer: a biomarker for antihistamine-resistant chronic urticaria.J Allergy Clin Immunol. 2013; 132: 983-986Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, plasma D-dimer concentrations are known to be rather nonspecific as they are known to be elevated in various inflammatory processes. A good biomarker in CSU should be elevated (only) in CSU patients and correlate with disease activity. As of yet, no good biomarker has been reported and confirmed by independent studies (Metz et al., 2013Metz M. Krull C. Maurer M. Histamine, TNF, C5a, IL-6, -9, -18, -31, -33, TSLP, neopterin, and VEGF are not elevated in chronic spontaneous urticaria.J Dermatol Sci. 2013; 70: 222-225Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We hypothesized that Substance P (SP), an 11-amino-acid peptide that acts primarily via the G-protein-coupled receptor neurokinin-1, could be both a biomarker and a factor involved in the pathogenesis of CSU since (1) at high concentrations, SP can induce the degranulation of mast cells in vitro (Kulka et al., 2008Kulka M. Sheen C.H. Tancowny B.P. et al.Neuropeptides activate human mast cell degranulation and chemokine production.Immunology. 2008; 123: 398-410Crossref PubMed Scopus (322) Google Scholar); (2) SP induces a wheal and flare response in vivo, which is more pronounced in patients with CSU as compared to healthy controls (Borici-Mazi et al., 1999Borici-Mazi R. Kouridakis S. Kontou-Fili K. Cutaneous responses to substance P and calcitonin gene-related peptide in chronic urticaria: the effect of cetirizine and dimethindene.Allergy. 1999; 54: 46-56Crossref PubMed Scopus (51) Google Scholar); and (3) low concentrations of SP, ie, concentrations that are likely to occur in the skin of patients, can increase the responsiveness of mast cells to activating signals (Janiszewski et al., 1994Janiszewski J. Bienenstock J. Blennerhassett M.G. Picomolar doses of substance P trigger electrical responses in mast cells without degranulation.Am J Physiol. 1994; 267: C138-C145PubMed Google Scholar; Forsythe and Bienenstock, 2012Forsythe P. Bienenstock J. The mast cell-nerve functional unit: a key component of physiologic and pathophysiologic responses.Chem Immunol Allergy. 2012; 98: 196-221Crossref PubMed Scopus (9) Google Scholar). To assess whether SP is a good biomarker in CSU, we first compared serum concentrations of SP in samples from 30 healthy subjects with 118 CSU patients from two independent urticaria clinics in Germany and observed a significant and more than fourfold increase in SP levels in patients with CSU (491±24 pg ml−1) compared to healthy controls (105±28 pg ml−1, P<0.0001, Figure 1a). Additionally, we have assessed SP levels in the serum of 20 patients with cold urticaria. Here, SP levels are also increased (280.3±24 pg ml−1), but significantly lower than in CSU patients (Figure 1a). All analyses have been carried out in adherence to the Helsinki Guidelines and after institutional approval with written and informed patient consent. Next, using the UAS7 as activity marker, we found a significant correlation between SP and disease activity (Figure 1b). Furthermore, very high levels of SP (>800 pg ml−1) were absent in healthy subjects and in mild CSU but found in every fourth patient with severe CSU (26%), while low levels of SP were predominant in healthy controls (77%) and rarely found in severe CSU patients (13%, Figure 1c). It has been shown that mast cells can be a source of SP (Katsuno et al., 2003Katsuno M. Aihara M. Kojima M. et al.Neuropeptides concentrations in the skin of a murine (NC/Nga mice) model of atopic dermatitis.J Dermatol Sci. 2003; 33: 55-65Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar); hence it can be speculated that elevated SP is due to massive mast cell degranulation in patients with high UAS. To address this possibility, we have correlated SP level with urticaria activity at the day of blood withdrawal, which did not reveal significant differences (Figure 1d), indicating that elevated SP levels in CSU patients are not due to SP released from mast cells. It has been hypothesized that SP is involved in the pathogenesis of angioedema in CSU (Akcali et al., 2008Akcali C. Ozkur M. Erbagci Z. et al.Association of insertion/deletion polymorphism of the angiotensin-converting enzyme gene with angio-oedema accompanying chronic urticaria but not chronic urticaria without angio-oedema or the autologous serum skin test response.J Eur Acad Dermatol Venereol. 2008; 22: 83-86PubMed Google Scholar). We, therefore, compared patients with (n=48) or without (n=11) angioedema and did not detect differences in SP levels between these groups (Figure 2a). Furthermore, comparison of SP levels among CSU patients based on the underlying etiology of CSU showed no differences between these groups (Figure 2b). CSU patients have a high prevalence of psychosomatic comorbidity and often report exacerbation of disease under stress (Staubach et al., 2006Staubach P. Eckhardt-Henn A. Dechene M. et al.Quality of life in patients with chronic urticaria is differentially impaired and determined by psychiatric comorbidity.Br J Dermatol. 2006; 154: 294-298Crossref PubMed Scopus (195) Google Scholar). As SP has been implicated not only in inflammatory diseases but also in the pathophysiology of depression and anxiety (Rosenkranz, 2007Rosenkranz M.A. Substance P at the nexus of mind and body in chronic inflammation and affective disorders.Psychol Bull. 2007; 133: 1007-1037Crossref PubMed Scopus (70) Google Scholar); we next examined SP in patients with or without increased levels of anxiety, depressive mood, or history of exacerbation of disease in stress. In the investigated population, we did not observe differences in SP between either group (Figure 2c and d). To rule out other potential bias, we also correlated SP levels with duration of disease and with the age and sex of the CSU patients. As expected, no correlation was observed for any of these groups (data not shown). Here, we show that SP is increased in CSU patients and that only disease activity but not underlying cause of disease, occurrence of angioedema, or psychosomatic comorbidities are correlated with serum concentrations of SP. Whether SP also contributes to the pathophysiology of CSU is as of yet unclear and cannot be answered by our findings. However, hints from the literature allow us to hypothesize about a potential role of SP: Unlike human skin mast cells, mucosal mast cells in the intestine do not constitutively express neurokinin-1, the main receptor for SP (Bischoff et al., 2004Bischoff S.C. Schwengberg S. Lorentz A. et al.Substance P and other neuropeptides do not induce mediator release in isolated human intestinal mast cells.Neurogastroenterol Motil. 2004; 16: 185-193Crossref PubMed Scopus (69) Google Scholar). Urticaria symptoms are mainly restricted to the skin, and despite the abundance of mast cells in the intestine urticaria does usually not affect the gastrointestinal tract. A role for SP in urticaria could explain this phenomenon. Furthermore, SP has been shown to act as a sensitizer for mast cells, i.e., increased SP levels may reduce the activation threshold of mast cells (Janiszewski et al., 1994Janiszewski J. Bienenstock J. Blennerhassett M.G. Picomolar doses of substance P trigger electrical responses in mast cells without degranulation.Am J Physiol. 1994; 267: C138-C145PubMed Google Scholar; Zuberbier et al., 2002Zuberbier T. Pfrommer C. Specht K. et al.Aromatic components of food as novel eliciting factors of pseudoallergic reactions in chronic urticaria.J Allergy Clin Immunol. 2002; 109: 343-348Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar; Forsythe and Bienenstock, 2012Forsythe P. Bienenstock J. The mast cell-nerve functional unit: a key component of physiologic and pathophysiologic responses.Chem Immunol Allergy. 2012; 98: 196-221Crossref PubMed Scopus (9) Google Scholar). Here, we have identified elevated SP regardless of the underlying cause of urticaria. We could thus hypothesize that elevated SP may be a unifying underlying sensitizer enabling other factors (e.g., complement, pseudoallergens, autoreactive substances) to reach threshold levels for activating mast cells. In a previous report, SP has not been found to be elevated in CSU (Tedeschi et al., 2005Tedeschi A. Lorini M. Asero R. No evidence of increased serum substance P levels in chronic urticaria patients with and without demonstrable circulating vasoactive factors.Clin Exp Dermatol. 2005; 30: 171-175Crossref PubMed Scopus (26) Google Scholar). In our hands there was a very robust and reproducible upregulation of SP in CSU patients from two independent centres as compared to healthy controls; we can therefore not explain this discrepancy. However, it has been shown before that bovine SP is markedly reduced in blood samples processed within 1 hour because of rapid degradation by enzymes (Mosher et al., 2014Mosher R.A. Coetzee J.F. Allen P.S. et al.Effects of sample handling methods on substance P concentrations and immunoreactivity in bovine blood samples.Am J Vet Res. 2014; 75: 109-116Crossref PubMed Scopus (15) Google Scholar). To avoid degradation of SP, we have processed blood within 30 minutes and stored serum at -80 °C. Possibly, differences in the blood processing have resulted in the observed discrepancies. Furthermore, we compared SP levels in serum with or without the addition of the enzyme inhibitor aprotinin and EDTA and did not observe differences in SP concentrations (data not shown). Taken together, our results indicate that SP has the potential to be used as a biomarker in CSU and that it could be a therapeutic target in CSU treatment. We thank Hesna Gözlükaya and Nikki Rooks for excellent patient care and management and Marina Frömming for technical assistance.