0 Aeolus Press Ocular Immunology and Inflammation 0927-3948/93/US$3.50 (Accepted 10 November 1992) Immunopathology of ocular onchocerciasis 3. Th-2 helper T cells in the conjunctivao CHI-CHAO CHAN*, QIAN LI, ANTOINE P. BREZIN, SCOTT M. WHITCUP, CHARLES EGWUAGU, ERIC A. OTTESON' and ROBERT B. NUSSENBLATT Laboratory of Immunology, National Eye Institute and 'Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA ABSTRACT. Ocular onchocerciasis is the second most common infectious cause of blindness in the world. Th-2 helper T (Th-2) cells are thought to play a critical role in immediate hypersensitivi- ty against allergens and extra-cellular parasitic infections. Th-2 cells secrete a high amount of IL-4, regulate IgE production, and recruit eosinophils and mast cells. Conjunctival biopsies from ten African patients with ocular onchocerciasis were evaluated for the presence of 11-2, IL-4, mast cells and major basic protein (MBP), a marker for eosinophils. IL-4 mRNA was detected in seven of ten conjunctival specimens using in situ hybridization, yet IL-4 was detected in only one specimen using immunohistochemical staining. In contrast, IL-2 mRNA was detected in three of ten conjunctival specimens and IL-2 was detected in two specimens. There were greater numbers of mast cells and the presence of MBP in specimens with IL-4 mRNA. Furthermore, the three biopsies containing both IL-2 and IL-4 mRNA had greater numbers of CD4+ cell infiltration and the patient with IL-4 protein in his conjunctiva also had the highest IgE in his aqueous humor. These findings suggest that Th-2 cells and their lymphokines are important for the localized host responsiveness to ocular onchocerciasis. Key words: onchocerciasis ; Th-1 helper cell ; Th-2 helper cell ; IL-2; IL-4 INTRODUCTION Ocular onchocerciasis is a common cause of blindness in the world, especially in western equatorial Africa, Central and South America. Approximately 86 million people live in endemic * Correspondence to: Chi-Chao Chan, M.D., Bldg. 10, Rm. 10N206, NIH/NEI, Bethesda, MD 20892, USA. Tel: (301)496-1243. Fax : (301)402-0485. O Presented at the Second International Symposium on Ocular Inflammation, Jerusalem, Israel, 30 August-3 Sep- tember, 1992. areas; 18 million people are infected and 340,000 are blind*-3. Onchocerciasis is caused by the tissue-dwelling filarial parasite Onchocerca volvulus. The infection is transmitted to man by black flies of a number of Simulium species. Blindness may result from keratitis, uveitis, secondary glaucoma, chorioretinal degeneration and optic nerve a t r ~ p h y ~ - ~ , although micro- filariae may be found in any part of the eye213. Recently, we have studied conjunctival, aque- ous humor and serum specimens from patients with chronic, active ocular onchocerciasis Ocular Immunology and Inflammation - 1993, Vol I, No. 1/2, pp. 71 - 77 0 Aeolus Press Buren (The Netherlands) 1993 71 Ocul Immunol Inflamm Downloaded from informahealthcare.com by National Library of Medicine on 07/08/14 For personal use only.
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0 Aeolus Press Ocular Immunology and Inflammation 0927-3948/93/US$3.50 (Accepted 10 November 1992)
Immunopathology of ocular onchocerciasis
3. Th-2 helper T cells in the conjunctivao
CHI-CHAO CHAN*, QIAN LI, ANTOINE P. BREZIN, SCOTT M. WHITCUP, CHARLES EGWUAGU, ERIC A. OTTESON' and ROBERT B. NUSSENBLATT
Laboratory of Immunology, National Eye Institute and 'Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA
ABSTRACT. Ocular onchocerciasis is the second most common infectious cause of blindness in the world. Th-2 helper T (Th-2) cells are thought to play a critical role in immediate hypersensitivi- ty against allergens and extra-cellular parasitic infections. Th-2 cells secrete a high amount of IL-4, regulate IgE production, and recruit eosinophils and mast cells. Conjunctival biopsies from ten African patients with ocular onchocerciasis were evaluated for the presence of 11-2, IL-4, mast cells and major basic protein (MBP), a marker for eosinophils. IL-4 mRNA was detected in seven of ten conjunctival specimens using in situ hybridization, yet IL-4 was detected in only one specimen using immunohistochemical staining. In contrast, IL-2 mRNA was detected in three of ten conjunctival specimens and IL-2 was detected in two specimens. There were greater numbers of mast cells and the presence of MBP in specimens with IL-4 mRNA. Furthermore, the three biopsies containing both IL-2 and IL-4 mRNA had greater numbers of CD4+ cell infiltration and the patient with IL-4 protein in his conjunctiva also had the highest IgE in his aqueous humor. These findings suggest that Th-2 cells and their lymphokines are important for the localized host responsiveness to ocular onchocerciasis.
Ocular onchocerciasis is a common cause of blindness in the world, especially in western equatorial Africa, Central and South America. Approximately 86 million people live in endemic
* Correspondence to: Chi-Chao Chan, M.D., Bldg. 10, Rm. 10N206, NIH/NEI, Bethesda, MD 20892, USA. Tel: (301)496-1243. Fax : (301)402-0485.
O Presented at the Second International Symposium on Ocular Inflammation, Jerusalem, Israel, 30 August-3 Sep- tember, 1992.
areas; 18 million people are infected and 340,000 are blind*-3. Onchocerciasis is caused by the tissue-dwelling filarial parasite Onchocerca volvulus. The infection is transmitted to man by black flies of a number of Simulium species. Blindness may result from keratitis, uveitis, secondary glaucoma, chorioretinal degeneration and optic nerve a t r ~ p h y ~ - ~ , although micro- filariae may be found in any part of the eye213.
Recently, we have studied conjunctival, aque- ous humor and serum specimens from patients with chronic, active ocular onchocerciasis
Ocular Immunology and Inflammation - 1993, Vol I , No. 1/2, pp. 71 - 77 0 Aeolus Press Buren (The Netherlands) 1993
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undergoing cataract extraction from the area of Tamale in northern Ghana5.6. Our previous findings of T lymphocytic infiltration and ocu- lar resident cellular activation showed the poten- tial importance of T cell-mediated immune process in the conjunctiva.
Two types of T helper cells, Th-1 and Th-2, have been defined based on their lymphokine production and immune response7,*. Th-1 cells preferentially secrete interleukine (IL-2) and participate in delayed type hypersensitivity. In contrast, Th-2 cells secrete high amounts of IL-4 and foster immediate hypersensitivity. In the present study, the ten conjunctival specimens5 from patients with ocular onchocerciasis were re-examined for the presence of IL-2 and IL-4 as an indication of the presence of Th-1 and Th-2 cells.
MATERIALS AND METHODS
Specimen collection
Snap frozen conjunctival biopsies were obtained from ten African men with chronic, active ocu- lar onchocerciasis in Tamale, Ghana. They were transported to National Eye Institute, USA, for evaluation. The investigations were performed according to the guidelines of the Declaration of Helsinki. The detailed medical history, labora- tory data and surgical procedures were reported previously5. The frozen conjunctival specimens were serial-sectioned and examined for the pres- ence of IL-2 (mRNA and protein), IL-4 (mRNA and protein), mast cells, eosinophils and major basic protein (MBP), a degranulation product of eosinophils.
Aqueous humor fluid samples and sera from these ten patients were also collected, frozen and sent to National Institutes of Allergy and Infec- tious Disease for total IgE assay.
Immunohistologic techniques
Cryostat sections of the conjunctival specimens were examined for the presence of IL-2 and IL-4 protein using avidin-biotin-immunoperoxidase (ABC) techniques previously described5. The primary antibodies were monoclonal anti IL-2 and polyclonal anti IL-4 antibody (Genzyme, Boston, MA), The secondary antibodies were biotinylated horse anti-mouse IgG (Vector Lab- oratory, Burlingame, CA) and biotinylated goat anti-rabbit IgG (Vector Laboratory, Burling- ame, CA) respectively. Slides were examined and graded by two independent observers (C-C.C. and S.M.W.).
Conjunctival specimens were examined for the presence of major basic protein (MBP) of the secondary granules of eosinophils using immunofluorescence technique (MBP is a gener- ous gift from Dr. Richard Davey, NIAID, NIH, Bethesda, MD). After staining, the slides were examined under Zeiss fluorescent microscopy and graded by two independent investigators (C- C. C. and Q. L.).
Toluidine blue staining was used to identify the mast cells in the conjunctiva. The slides were stained with 0.15% toluidine blue solution, cov- ered with gelvatol (Indian Orchard Plant, Springfield, MA), examined under Zeiss light microscope, and graded by two independent investigators (C-C. C. and Q. L.).
In situ hybridization
In situ hybridization was performed using non- radioactive digoxigenin labeled riboprobes7. 8.
The IL-4 cDNA fragment 373 bp (Hind III- EcoR1) and the IL-2 cDNA fragment 910 bp (Bam H1-Bam H1) were initially subcloned into the pBluescript I1 KS +/- vector. The mRNA sense and antisense probes were then prepared
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by the in vitro transcription with digoxigenin- UTP (Dig RNA Labeling Kit, Boehringer Mannheim Biochemicals, Indianapolis, IN) and used for in situ hybridization on the conjuncti- val cryostat sections. Following overnight hybri- dization (37-42"C), and washing with graded SSC solution (sodium chloride, sodium citrate) and RNase treatment, immunodetection was conducted using Nucleic Acid Detection Kit (Boehringer Mannheim Biochemicals, Indiana- polis, IN). Slides were blocked with sheep serum, layered with anti-digoxigenin antibody (1 500) and incubated in a humid chamber for three hours at room temperature. After appro- priate washing, color solution containing NBT/ x-phosphate/Levamisole was applied overnight in a dark, humid chamber for the determination of color reaction. TE buffer was then added to terminate the reaction. Slides were covered with Gelvetol and stored at 4°C until they were grad- ed by three independent, masked observers (C- C.C.,Q.L.andS.M.W.).
IgE analysis
Total IgE in the aqueous humor and serum was measured by radioassay using the Prist Kit (Pharmacia, Piscataway, NY).
RESULTS
IL-2 and IL-4
IL-4 mRNA was detected in seven of ten con- junctival specimens by in situ hybridization (Fig. l), IL-4 protein was detected in only one of the ten specimens (Case 7) by immunohisto- chemistry (Table 1). In contrast, IL-2 mRNA was detected in three of ten conjunctival speci- mens (Fig. 2), and IL-2 protein was detected in two of these (Table 1). Three specimens contain-
ing both IL-2 mRNA and IL-4 mRNA had greater numbers of CD4 + cell infiltration (Table 1).
Mast cells
At least one mast cell per high power ( x 40) field was identified in each conjunctival biopsy speci- men (Table 2). Greater numbers of mast cells were found in the specimens with detectable IL- 4 mRNA and its protein. In addition, the two specimens that contained both IL-2 mRNA and protein showed the highest number of mast cell infiltration.
Eosinophils: major basic protein (MBP)
Few eosinophils were noted in these conjuncti- val biopsy specimens. However, MBP in the substantia propria was visualized in all biopsies (Table 2). Nevertheless, there was no correlation between the number of the infiltrating cells and the presence of lymphokines with the amount of MBP in the specimen.
Total IgE was detected in the aqueous humor in five of nine samples studied (one sample had insufficient quantity to be examined, Table 3). It is interesting that only one patient (Case 7) had a high ratio of aqueous humor to serum levels of IgE. This patient was the only patient who had detectable IL-4 protein in his conjunc- tival biopsy.
DISCUSSION
We previously documented the importance of T-cell mediated immune process on these ten conjunctival specimens'. The predominant
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Fig. 1. Micrograph of the conjunctival specimen from Case 7 showing expression of IL-4 mRNA. A. anti-sense probe. B. sense probe. ( x 400, reduced 35%).
infiltrating cells in the conjunctiva are T lym- phocytes (CD3 + cells) with equal numbers of T helper (CD4) and T suppressor (CD8) subtypes. Greater CD4 + cells are found in the conjuncti- va of the patients with ocular onchocerciasis
compared with non-onchocerciasis population ; however, the phenotypes and functions of the infiltrating CD4 cells have not been defined. The present study was designed to answer these questions by examining lymphokine secretion,
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Fig. 2. Micrograph of the conjunctival specimen from Case 7 showing expression of IL-2 mRNA. A. anti-sense probe. B. sense probe. ( x 400, reduced 35%).
mast cell number and MBP of the eosinophil in clones9. Th-1 clones produce IL-2, interferon-y the biopsy specimen as well as IgE in the eye. (IFN y) and lymphotoxin (LT); whereas Th-2
Two patterns of lymphokine secretion have clones secrete IL-4, IL-5, IL-6 and IL-lO'O. The initially been defined among mouse T-cell different cytokines produced by these cells are
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TABLE 1. CD4 + cells, IL-2 and IL-4 in conjunctival biop- sies of patients (n = 10) with ocular onchocerciasis
Case CD4+ * IL-2 IL-4 protein/mRNA protein/mRNA
TABLE 3. Total IgE in serum and aqueous humor of pa- tients (n = 10) with ocular ochocerciasis
TABLE 2. Mast cells and MBP (eosinophil degranulation products) in conjunctival biopsies of patients (n = 10) with ocular onchocerciasis
Case Mast cell* MBP (eosinophil)
1 2 3 4 5 6 7 8 9
10
10 8
29 I 1
12 30
1 1
10
+ + + + + + + + + +
thought to account for the different functions of the two types of T cells6. Although most human CD4+ T cell clones have the intrinsic capacity to produce IL-2, IFN y, IL-4, IL-5 and IL-10, major quantitative and occasionally qualitative differences in lymphokine production levels have been observed after different modes of activation7. 9 9 lo. The predominant lymphokine in the conjunctiva obtained from the ten patients with ocular onchocerciasis is IL-4, which is produced mainly by Th-2 and mast
cells6,7,9-12. This lymphokine can further stimu- late the proliferation, development and matura- tion of Th-2 cellsI3.
The infection of Onchocerca volvulus induces predominantly Th-2 cell responses. These responses result in significant IL-4 produc- tion7. l1-I6, eosinophil and mast cell infil- t r a t e~" - '~ in the conjunctiva, elevation of IgE15 in the aqueous humor and serum, and absence of delayed type hypersensitivity7.8. This immediate type immune reaction is further per- petuated by the products of eosinophils and mast cells, leading to the classical clinical symp- toms of itching, conjunctival injection, keratitis, and uveitis. Two of the three patients who were negative for IL-2 and IL-4 denied epiphora and itching. In contrast, three of the seven patients who were positive for these lym- phokines did not complain of epiphora and itch- ing.
A clear understanding of the immune response against parasitic infection is important for the development of optimal therapy. In the eye, however, the immune responses often lead to severe consequences and permanent loss of vision. Understanding the role of Th-2 cells and
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the elaborated lymphokines will allow us to reg- before, during and after treatment will have ulate the immune response to enhance killing of important implications for immunological inter- the parasite while simultaneously limiting the ventions that may be useful to treat parasitic inflammatory damage to the eye. Further stud- diseases such as ocular onchocerciasis, the ies on specimens obtained from onchocerciasis fourth leading cause of blindness in the world. patients with differing clinical manifestations
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