Identification of local Th2 and Th0 lymphocytes in vernal conjunctivitis by cytokine flow cytometry.
OBJECTIVE
Th2 lymphocytes may play a key role in the development of allergic diseases such as vernal keratoconjunctivitis (VKC). Cytokine flow cytometry of tear samples was used to identify the phenotypical and functional properties of lymphocytes at the actual site of the allergic reaction.


METHODS
Tear and blood samples were obtained from patients affected by active VKC (n = 12) and from normal control subjects (n = 10). Tears were obtained after gentle scraping of the tarsal and bulbar conjunctiva. Tear and blood samples were placed in a solution of brefeldin-A, phorbol myristate acetate (PMA), ionomycin, and RPMI for 4 hours and then processed for flow cytometry. Lymphocytes were marked with the monoclonal antibodies, anti-IFN-gamma and anti-interleukin (IL)-4. Levels of IL-4, IL-2, IFN-gamma, IL-2R, total IgE, eosinophil cationic protein (ECP), eosinophil protein X/neurotoxin (EPX), and myeloperoxidase (MPO) were also evaluated in serum.


RESULTS
Expression of IL-4 was observed in 9.2%+/-9.5% of lymphocytes in tears of patients with VKC. Of the 12 patients with VKC, 8 (67%) had tear lymphocytes positive for IL-4 (Th2). Two patients (17%) had a double population of lymphocytes: One was positive for Th2, and the other was positive for both IL-4 and IFN-gamma (Th0). One patient (8%) was positive for IFN-gamma (Th1) only, and one patient was negative for both ILs. No differences in the percentage of Th2 lymphocytes were found between tarsal and limbal patients. The percentage of Th2 lymphocytes was significantly correlated with the severity of the disease. No positive lymphocytes were found in tears of control subjects. Eosinophils, serum IgE, ECP, and EPX were all significantly higher in VKC than in control subjects.


CONCLUSIONS
In ocular allergic diseases, local lymphocytes expressed the Th2 phenotype and, to a lesser degree, the Th0 phenotype. Although results of systemic allergic markers can be inconclusive in patients with VKC, flow cytometry demonstrated a local lymphocyte phenotype that can account for the clinical and histologic abnormalities of VKC.
Leonardi
A
A
Department of Physiopathologic Optics, Institute of Ophthalmology, University of Padua, Italy. mdvol@tin.it
DeFranchis
G
G
Zancanaro
F
F
Crivellari
G
G
De Paoli
M
M
Plebani
M
M
Secchi
A G
AG
D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002648:Child; D003233:Conjunctivitis, Allergic / Q000097:blood / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000032:analysis*; D015847:Interleukin-4 / Q000032:analysis*; D008297:Male; D013666:Tears / Q000737:chemistry; D018418:Th2 Cells / Q000276:immunology*
D015847:Interleukin-4; D007371:Interferon-gamma