- Email: [email protected]
Altered expression of chemoattractant receptor–homologous molecule expressed on TH2 cells on blood basophils and eosinophils in patients with chronic spontaneous urticaria
Letter to the Editor Altered expression of chemoattractant receptor–homologous molecule expressed on TH2 cells on blood basophils and eosinophils in patients with chronic spontaneous urticaria To the Editor: Chronic spontaneous urticaria (CSU) has a significant effect on patients’ quality of life through symptoms of pruritus and recurrent skin lesions. Biopsies of CSU skin lesions consistently reveal degranulated mast cells and infiltration by leukocytes, such as basophils, eosinophils, and T lymphocytes.1 A putative role for basophils in patients with CSU is supported by the association of disease severity with basopenia and the selective accumulation of basophils in CSU skin lesions.1 A study of the biopsies of 24 skin diseases revealed that CSU lesions had the highest numbers of infiltrating basophils and equal numbers of infiltrating eosinophils, implicating both cells in disease pathology.2 Because bone marrow production of basophils is normal in patients with CSU,3 the blood basopenia seen in these patients appears to be the result of potent recruitment of basophils to the skin. Blood basophils express the chemokine receptors CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR2, and CXCR4. Relative to healthy subjects, increased serum levels of CCL5/RANTES
(binds CCR1, CCR3, and CCR5) and eotaxin (binds CCR3) are reported in patients with CSU.4,5 Given the increased serum chemokine levels, basophil infiltration of the skin, and basopenia in patients with CSU, we examined chemokine receptor levels on blood basophils in these groups. In addition, prostaglandin D2 (PGD2), a product of activated mast cells, is known to cause activation and chemotaxis of basophils and eosinophils through its receptor, chemoattractant receptor–homologous molecule expressed on TH2 cells (CRTH2). Although the exact role of PGD2 in patients with CSU is unknown, polymorphisms in the CRTH2 promoter region are associated with antihistamine-resistant CSU.6 On the basis of these observations, we also examined CRTH2 expression on basophils and eosinophils of patients with CSU relative to that seen in nonallergic control subjects. We recruited patients with active CSU and nonallergic adults at the Johns Hopkins Asthma and Allergy Center, as approved by the Johns Hopkins Hospital Institutional Review Board and Western Institutional Review Board. The diagnosis of CSU was determined by Johns Hopkins Asthma and Allergy Center allergists based on established criteria.1 Subjects were excluded if they had taken oral steroids, sulfasalazine, dapsone, omalizumab, or immunosuppressants within 30 days. Healthy control subjects lacked histories of asthma, eczema, or allergic rhinitis.
FIG 1. Blood basophil and eosinophil chemoattractant receptor expression. A and B, CRTH2 expression for freshly isolated basophils (Fig 1, A) and eosinophils (Fig 1, B) from patients with CSU and healthy control subjects. 1P < .05. C, Basophil chemokine receptor expression for patients with CSU (n 5 23) and healthy control subjects (n 5 8). D, Basophil CRTH2 expression for 1 healthy control subject after PGD2 stimulation for 15 minutes (representative of 3 experiments). MFI, Mean fluorescence intensity.
1
2 LETTER TO THE EDITOR
Net ScaƩer Value
CSU 50 45 40 35 30 25 20 15 10 5 0
J ALLERGY CLIN IMMUNOL nnn 2015
non-allergic controls
+
-8.0
-7.5
-7.0 -6.5 [PGD2] [log10 M]
-6.0
-5.0
FIG 2. PGD2-induced average eosinophil shape change, as determined by net scatter movement, is shown for patients with CSU (n 5 15) and healthy control subjects (n 5 7). Error bars are representative of the SD. 1P < .05.
After obtaining informed consent, whole blood samples were collected in heparin Vacutainer tubes (BD, Franklin Lakes, NJ), washed in calcium free buffer, and examined with allophycocyanin-CD123, the appropriate IgG controls, or receptor antibodies against CRTH2 (Miltenyi Biotec, Bergisch Gladbach, Germany), CCR1 (Miltenyi Biotec), CCR2 (Novus Biologicals, Littleton, Colo), CCR3 (eBioscience, San Diego, Calif), CCR5 (eBioscience), CXCR1 (Novus Biologicals), or CD16 (Beckman Coulter, Fullerton, Calif). Aliquots of whole blood were incubated with PGD2 (P5172, Sigma, St Louis, Mo; 1028 to 1025 mol/L) or buffer for 15 minutes at 378C and then labeled for flow cytometry to determine the effects of PGD2 stimulation on eosinophil shape change and basophil CRTH2 surface expression. Erythrocytes were lysed with a whole blood lysing reagent kit (Beckman Coulter), and leukocytes were suspended in 1% paraformaldehyde. For eosinophil shape change, cell movement was assessed by using forward/side scatter with net cellular movement recorded as follows: Value at PGD2 concentration 2 Value with buffer alone. Samples were run on a BD FACSCalibur flow cytometer. The Mann-Whitney U test was used for baseline receptor comparisons between patients with CSU and healthy control subjects. Prism software (version 6.00; GraphPad Software, La Jolla, Calif) was used for statistical analysis. A P value of less than .05 was considered significant. We enrolled a total of 31 subjects (23 patients with CSU and 8 nonallergic control subjects) for basophil receptor expression (see Table E1 in this article’s Online Repository at www.jacionline. org). A smaller subset were also examined for eosinophil studies. The majority of subjects were female and white. All patients with CSU were taking approved doses of H1-receptor antagonists. No subjects were taking leukotriene receptor antagonists or doxepin. We found that surface expression of CRTH2 was significantly lower on blood basophils (mean fluorescence intensity, 300.2 vs 398.4; P 5 .0178) and eosinophils (mean fluorescence intensity, 60.27 vs 70.88; P 5 .0439) in patients with CSU compared with levels in healthy control subjects (Fig 1, A and B). In contrast, there was no significant difference in levels of the other candidate basophil chemoattractant surface receptors (Fig 1, C), including CCR5 or CXCR3 (data not shown). We confirmed that PGD2 stimulation of healthy basophils at doses greater than 1026.5 mol/L decreased CRTH2 surface expression (Fig 1, D).
In addition, ex vivo PGD2-induced eosinophil shape change of patients with CSU versus healthy control subjects was modestly reduced across all concentrations of PGD2 (Fig 2), which is consistent with receptor desensitization. Unique features of CSU include blood basophil migration to skin lesions and basopenia, but the recruitment pathways for basophil migration to the skin are unknown. Although increased serum levels of both RANTES and eotaxin are reported in patients with CSU,4,5 we found that expression levels of the corresponding basophil chemokine surface receptors were similar between patients with active CSU and control subjects. Biopsy specimens of hives from patients with CSU demonstrate mast cell degranulation and infiltration by basophils, eosinophils, and T lymphocytes,1 all of which express CRTH2. Our findings of reduced surface expression of CRTH2 on blood basophils and eosinophils in patients with CSU are consistent with ongoing PGD2 exposure in vivo. CRTH2 is internalized on eosinophils after PGD2 binding.7 We have demonstrated for the first time that PGD2 stimulation induces dosedependent CRTH2 receptor internalization on human basophils. We further observed that ex vivo PGD2-induced eosinophil shape change was less robust among patients with CSU. Thus it is plausible that decreased CRTH2 levels on basophils and eosinophils in patients with CSU are due to in vivo PGD2 activation, suggesting engagement of CRTH2 in patients with CSU. A previous study reported increased CRTH2 expression on blood eosinophils in patients with chronic urticaria, although in the absence of skin symptoms.8 The clinical consequences of CRTH2 activation in patients with CSU remain unknown. In clinical trials, oral CRTH2 antagonists have provided symptomatic improvements in patients with allergic asthma and rhinoconjunctivitis.9 Further studies are needed to determine the role of the PGD2/CRTH2 pathway in selective migration of basophils and eosinophils into skin lesions of patients with CSU and whether CRTH2 antagonists are effective for CSU disease management. Eric Tyrell Oliver, MD Patricia Meghan Sterba, MS Kelly Devine, BS Becky M. Vonakis, PhD Sarbjit Singh Saini, MD From the Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Md. E-mail: [email protected]. Supported through self-funds and AstraZeneca. AstraZeneca had no involvement in the design of this study, data collection, analysis or interpretation of the results, writing of this report, or decision to submit this report for publication. Disclosure of potential conflict of interest: S. S. Saini has received research support from AstraZeneca, Novartis, the National Institutes of Health/National Institute of Allergy and Infectious Diseases, and ITN; has received consultancy fees from Ono, Novartis, Genentech, and Medimmune; and has received royalties from UpToDate. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Saini SS. Chronic spontaneous urticaria: etiology and pathogenesis. Immunol Allergy Clin North Am 2014;34:33-52. 2. Ito Y, Satoh T, Takayama K, Miyagishi C, Walls AF, Yokozeki H. Basophil recruitment and activation in inflammatory skin diseases. Allergy 2011;66:1107-13. 3. Rorsman H. Basopenia in urticaria. Acta Allergol 1961;16:185-215. 4. Tedeschi A, Asero R, Lorini M, Marzano AV, Cugno M. Serum eotaxin levels in patients with chronic spontaneous urticaria. Eur Ann Allergy Clin Immunol 2012;44:188-92. 5. Puxeddu I, Panza F, Pratesi F, Bartaloni D, Casigliani Rabl S, Rocchi V, et al. CCL5/RANTES, sVCAM-1, and sICAM-1 in chronic spontaneous urticaria. Int Arch Allergy Immunol 2013;162:330-4.
J ALLERGY CLIN IMMUNOL VOLUME nnn, NUMBER nn
6. Palikhe NS, Kim SH, Ye YM, Hur GY, Cho BY, Park HS. Association of CRTH2 gene polymorphisms with the required dose of antihistamines in patients with chronic urticaria. Pharmacogenomics 2009;10: 375-83. 7. Hamada K, Yamada Y, Kamada Y, Ueki S, Yamaguchi K, Oyamada H, et al. Prostaglandin D2 and interleukin-5 reduce CRTH2 surface expression on human eosinophils. Allergol Int 2004;53:179-84.
LETTER TO THE EDITOR 3
8. Yahara H, Satoh T, Miyagishi C, Yokozeki H. Increased expression of CRTH2 on eosinophils in allergic skin diseases. J Eur Acad Dermatol Venereol 2010;24:75-6. 9. Diamant Z, Sidharta PN, Singh D, O’Connor BJ, Zuiker R, Leaker BR, et al. Setipiprant, a selective CRTH2 antagonist, reduces allergen-induced airway responses in allergic asthmatics. Clin Exp Allergy 2014;44:1044-52. http://dx.doi.org/10.1016/j.jaci.2015.06.004
3.e1 LETTER TO THE EDITOR
J ALLERGY CLIN IMMUNOL nnn 2015
TABLE E1. Subjects’ characteristics
Age (y) Female sex Race/ethnicity White African American Asian Hispanic Pacific Islander Medication use Antihistamines (H1 6 H2) Leukotriene receptor antagonists Tricyclic antidepressants Thyroid replacement therapy Oral corticosteroids Disease duration 6 mo-1 y 1-2 y 2-3 y 3-4 y >4 y
Patients with CSU (n 5 23)
Nonallergic subjects (n 5 8)
43 (23-70) 68.4
34.5 (22-54) 62.5
60.8 13 13 8.7 4.3
75 12.5 12.5 0 0
100 0 0 4.3 0 13 8.7 4.3 4.3 69.6
Values are expressed as percentages or medians (ranges).
(binds CCR1, CCR3, and CCR5) and eotaxin (binds CCR3) are reported in patients with CSU.4,5 Given the increased serum chemokine levels, basophil infiltration of the skin, and basopenia in patients with CSU, we examined chemokine receptor levels on blood basophils in these groups. In addition, prostaglandin D2 (PGD2), a product of activated mast cells, is known to cause activation and chemotaxis of basophils and eosinophils through its receptor, chemoattractant receptor–homologous molecule expressed on TH2 cells (CRTH2). Although the exact role of PGD2 in patients with CSU is unknown, polymorphisms in the CRTH2 promoter region are associated with antihistamine-resistant CSU.6 On the basis of these observations, we also examined CRTH2 expression on basophils and eosinophils of patients with CSU relative to that seen in nonallergic control subjects. We recruited patients with active CSU and nonallergic adults at the Johns Hopkins Asthma and Allergy Center, as approved by the Johns Hopkins Hospital Institutional Review Board and Western Institutional Review Board. The diagnosis of CSU was determined by Johns Hopkins Asthma and Allergy Center allergists based on established criteria.1 Subjects were excluded if they had taken oral steroids, sulfasalazine, dapsone, omalizumab, or immunosuppressants within 30 days. Healthy control subjects lacked histories of asthma, eczema, or allergic rhinitis.
FIG 1. Blood basophil and eosinophil chemoattractant receptor expression. A and B, CRTH2 expression for freshly isolated basophils (Fig 1, A) and eosinophils (Fig 1, B) from patients with CSU and healthy control subjects. 1P < .05. C, Basophil chemokine receptor expression for patients with CSU (n 5 23) and healthy control subjects (n 5 8). D, Basophil CRTH2 expression for 1 healthy control subject after PGD2 stimulation for 15 minutes (representative of 3 experiments). MFI, Mean fluorescence intensity.
1
2 LETTER TO THE EDITOR
Net ScaƩer Value
CSU 50 45 40 35 30 25 20 15 10 5 0
J ALLERGY CLIN IMMUNOL nnn 2015
non-allergic controls
+
-8.0
-7.5
-7.0 -6.5 [PGD2] [log10 M]
-6.0
-5.0
FIG 2. PGD2-induced average eosinophil shape change, as determined by net scatter movement, is shown for patients with CSU (n 5 15) and healthy control subjects (n 5 7). Error bars are representative of the SD. 1P < .05.
After obtaining informed consent, whole blood samples were collected in heparin Vacutainer tubes (BD, Franklin Lakes, NJ), washed in calcium free buffer, and examined with allophycocyanin-CD123, the appropriate IgG controls, or receptor antibodies against CRTH2 (Miltenyi Biotec, Bergisch Gladbach, Germany), CCR1 (Miltenyi Biotec), CCR2 (Novus Biologicals, Littleton, Colo), CCR3 (eBioscience, San Diego, Calif), CCR5 (eBioscience), CXCR1 (Novus Biologicals), or CD16 (Beckman Coulter, Fullerton, Calif). Aliquots of whole blood were incubated with PGD2 (P5172, Sigma, St Louis, Mo; 1028 to 1025 mol/L) or buffer for 15 minutes at 378C and then labeled for flow cytometry to determine the effects of PGD2 stimulation on eosinophil shape change and basophil CRTH2 surface expression. Erythrocytes were lysed with a whole blood lysing reagent kit (Beckman Coulter), and leukocytes were suspended in 1% paraformaldehyde. For eosinophil shape change, cell movement was assessed by using forward/side scatter with net cellular movement recorded as follows: Value at PGD2 concentration 2 Value with buffer alone. Samples were run on a BD FACSCalibur flow cytometer. The Mann-Whitney U test was used for baseline receptor comparisons between patients with CSU and healthy control subjects. Prism software (version 6.00; GraphPad Software, La Jolla, Calif) was used for statistical analysis. A P value of less than .05 was considered significant. We enrolled a total of 31 subjects (23 patients with CSU and 8 nonallergic control subjects) for basophil receptor expression (see Table E1 in this article’s Online Repository at www.jacionline. org). A smaller subset were also examined for eosinophil studies. The majority of subjects were female and white. All patients with CSU were taking approved doses of H1-receptor antagonists. No subjects were taking leukotriene receptor antagonists or doxepin. We found that surface expression of CRTH2 was significantly lower on blood basophils (mean fluorescence intensity, 300.2 vs 398.4; P 5 .0178) and eosinophils (mean fluorescence intensity, 60.27 vs 70.88; P 5 .0439) in patients with CSU compared with levels in healthy control subjects (Fig 1, A and B). In contrast, there was no significant difference in levels of the other candidate basophil chemoattractant surface receptors (Fig 1, C), including CCR5 or CXCR3 (data not shown). We confirmed that PGD2 stimulation of healthy basophils at doses greater than 1026.5 mol/L decreased CRTH2 surface expression (Fig 1, D).
In addition, ex vivo PGD2-induced eosinophil shape change of patients with CSU versus healthy control subjects was modestly reduced across all concentrations of PGD2 (Fig 2), which is consistent with receptor desensitization. Unique features of CSU include blood basophil migration to skin lesions and basopenia, but the recruitment pathways for basophil migration to the skin are unknown. Although increased serum levels of both RANTES and eotaxin are reported in patients with CSU,4,5 we found that expression levels of the corresponding basophil chemokine surface receptors were similar between patients with active CSU and control subjects. Biopsy specimens of hives from patients with CSU demonstrate mast cell degranulation and infiltration by basophils, eosinophils, and T lymphocytes,1 all of which express CRTH2. Our findings of reduced surface expression of CRTH2 on blood basophils and eosinophils in patients with CSU are consistent with ongoing PGD2 exposure in vivo. CRTH2 is internalized on eosinophils after PGD2 binding.7 We have demonstrated for the first time that PGD2 stimulation induces dosedependent CRTH2 receptor internalization on human basophils. We further observed that ex vivo PGD2-induced eosinophil shape change was less robust among patients with CSU. Thus it is plausible that decreased CRTH2 levels on basophils and eosinophils in patients with CSU are due to in vivo PGD2 activation, suggesting engagement of CRTH2 in patients with CSU. A previous study reported increased CRTH2 expression on blood eosinophils in patients with chronic urticaria, although in the absence of skin symptoms.8 The clinical consequences of CRTH2 activation in patients with CSU remain unknown. In clinical trials, oral CRTH2 antagonists have provided symptomatic improvements in patients with allergic asthma and rhinoconjunctivitis.9 Further studies are needed to determine the role of the PGD2/CRTH2 pathway in selective migration of basophils and eosinophils into skin lesions of patients with CSU and whether CRTH2 antagonists are effective for CSU disease management. Eric Tyrell Oliver, MD Patricia Meghan Sterba, MS Kelly Devine, BS Becky M. Vonakis, PhD Sarbjit Singh Saini, MD From the Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Md. E-mail: [email protected]. Supported through self-funds and AstraZeneca. AstraZeneca had no involvement in the design of this study, data collection, analysis or interpretation of the results, writing of this report, or decision to submit this report for publication. Disclosure of potential conflict of interest: S. S. Saini has received research support from AstraZeneca, Novartis, the National Institutes of Health/National Institute of Allergy and Infectious Diseases, and ITN; has received consultancy fees from Ono, Novartis, Genentech, and Medimmune; and has received royalties from UpToDate. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Saini SS. Chronic spontaneous urticaria: etiology and pathogenesis. Immunol Allergy Clin North Am 2014;34:33-52. 2. Ito Y, Satoh T, Takayama K, Miyagishi C, Walls AF, Yokozeki H. Basophil recruitment and activation in inflammatory skin diseases. Allergy 2011;66:1107-13. 3. Rorsman H. Basopenia in urticaria. Acta Allergol 1961;16:185-215. 4. Tedeschi A, Asero R, Lorini M, Marzano AV, Cugno M. Serum eotaxin levels in patients with chronic spontaneous urticaria. Eur Ann Allergy Clin Immunol 2012;44:188-92. 5. Puxeddu I, Panza F, Pratesi F, Bartaloni D, Casigliani Rabl S, Rocchi V, et al. CCL5/RANTES, sVCAM-1, and sICAM-1 in chronic spontaneous urticaria. Int Arch Allergy Immunol 2013;162:330-4.
J ALLERGY CLIN IMMUNOL VOLUME nnn, NUMBER nn
6. Palikhe NS, Kim SH, Ye YM, Hur GY, Cho BY, Park HS. Association of CRTH2 gene polymorphisms with the required dose of antihistamines in patients with chronic urticaria. Pharmacogenomics 2009;10: 375-83. 7. Hamada K, Yamada Y, Kamada Y, Ueki S, Yamaguchi K, Oyamada H, et al. Prostaglandin D2 and interleukin-5 reduce CRTH2 surface expression on human eosinophils. Allergol Int 2004;53:179-84.
LETTER TO THE EDITOR 3
8. Yahara H, Satoh T, Miyagishi C, Yokozeki H. Increased expression of CRTH2 on eosinophils in allergic skin diseases. J Eur Acad Dermatol Venereol 2010;24:75-6. 9. Diamant Z, Sidharta PN, Singh D, O’Connor BJ, Zuiker R, Leaker BR, et al. Setipiprant, a selective CRTH2 antagonist, reduces allergen-induced airway responses in allergic asthmatics. Clin Exp Allergy 2014;44:1044-52. http://dx.doi.org/10.1016/j.jaci.2015.06.004
3.e1 LETTER TO THE EDITOR
J ALLERGY CLIN IMMUNOL nnn 2015
TABLE E1. Subjects’ characteristics
Age (y) Female sex Race/ethnicity White African American Asian Hispanic Pacific Islander Medication use Antihistamines (H1 6 H2) Leukotriene receptor antagonists Tricyclic antidepressants Thyroid replacement therapy Oral corticosteroids Disease duration 6 mo-1 y 1-2 y 2-3 y 3-4 y >4 y
Patients with CSU (n 5 23)
Nonallergic subjects (n 5 8)
43 (23-70) 68.4
34.5 (22-54) 62.5
60.8 13 13 8.7 4.3
75 12.5 12.5 0 0
100 0 0 4.3 0 13 8.7 4.3 4.3 69.6
Values are expressed as percentages or medians (ranges).