Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium

PURPOSE. To study the presence of muscarinic and ␣-and ␤-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line. METHODS. Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M 1 -, M 2 -, and M 3 -muscarinic and to ␣ 1A -, ␣ 1B -, ␣ 1D -, ␣ 2A -, ␣ 2B -, ␣ 2C -, ␤ 1 -, ␤ 2 -, and ␤ 3 -adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-␣ and/or interferon (IFN)-␥. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS. By immunofluorescence microscopy, all receptor subtypes, except the ␣ 2C -adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M 2 -and M 3 -muscarinic receptors and ␣ 1A -, ␣ 1B -, ␣ 1D -, ␣ 2A -, ␣ 2B -, ␣ 2C -, ␤ 1 -, and ␤ 3 -adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M 1 -muscarinic and ␤ 2 -adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M 2 and M 3 -muscarinic and ␣ 1A -, ␣ 2A -, ␣ 2B -, ␤ 1 -and ␤ 2 -adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M 2 -muscarinic and ␣ 1B -and ␣ 2B -adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN␥ and/or TNF␣. CONCLUSIONS. The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation. (Invest Ophthalmol Vis Sci. 2005;46:504 -513

NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3(+)CD103(+) (~22%), CD3(+)γδ(+) (~9.6%), CD3(+)NK(+) (2%), CD3(-)NK(+) (~4.4%), CD3(+)CD8α (~0.9%), and CD4 (~0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17(+) cells in the cervical lymph nodes and generation of less pathogenic CD4(+)T cells. B6.nude recipient mice of adoptively transferred CD4(+)T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4(+)T cells.Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response

Location of intra-epithelial lymphocytes in conjunctiva before and after desiccating stress.

<p>Representative digital pictures of immunohistochemical staining of CD4⁺, CD8α⁺, CD103⁺, γδTCR⁺, NK⁺ in the conjunctivae of nonstressed (NS) C57BL/6 mice and after desiccating stress for 5 or 10 days (DS5, DS10). Inset in γδTCR row shows γδTCR in skin, which was used as positive control. Original magnification 40X; scale bar 20 µm. Number insets represent cell counts in the goblet cell rich of the conjunctiva in immunostained tissue sections in conjunctival epithelium of C57BL/6 mice. Data represents mean ± SD of cells/mm. Experiments were repeated three times with two mice per group per experiment. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.01 comparison vs. NS control.</p

Cytokine burst from NK positive populations after DS.

<p>mRNA levels in NK/NKT positive (+) and NK/NKT negative (–) cells isolated from nonstressed (NS) spleen and ocular surface (OS) and at different time points after desiccating stress (DS; DS1 = DS for 1 day, DS5 = DS for 5 days, DS10 = DS10 for 10 days). Unfractionated spleen was used as calibrator. Experiments were repeated two times with at least three samples per group per experiment. Because the standard deviation is relatively small compared to the levels of IL-6, IL-23 and IL-17A expression, the error bars do not show in the graph. * indicates p<0.05, *** indicates p<0.001 comparison vs. NS control NK/NKT+. ^∧ indicates P<0.05, ^∧∧∧ indicates p<0.001 comparison vs. NS control NK/NKT−.</p

Flow cytometry analysis of freshly isolated cells from the ocular surface.

<p>Data is presented as mean ±standard deviation of 5–6 different experiments per group/time point. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”), subsequently gated based on forward scatter height vs. forward scatter area and propridium iodide live/dead exclusion (“live gated cells”). Data presented represents percentage of positive (⁺) or negative (^-) cells after background subtraction.</p><p>NS = non-stressed, DS5 = desiccating stress for 5 days, DS10 = desiccating stress for 10 days.</p

Adoptive transfer results.

<p><b>A</b> Mean± SD of IL-17 ELISPOTs showing IL-17- producing cells isolated from the ocular surface (OS) and CD4⁺ T cells isolated from spleen and cervical lymph nodes (CLN) in donor mice that received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) antibody before (non-stressed, NS) and after 5 days of desiccating stress (DS5). Experiments were repeated two times with at least five mice per group per experiment. <b>B</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>C.</b> Bar charts show mean ± SD of three independent experiments with five mice for each group per experiment. <b>D-G-</b> Laser scanning immunofluorescent confocal microscopy of cornea immunostained for MMP-3 (in <b>D</b>) and MMP-9 (in <b>F</b>) in nude mice that received CD4⁺T cells isolated from donor mice treated with systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Bar graphs are mean±SD of fluorescence intensity measured in corneal epithelium for MMP-3 (E) and MMP-9 (G) of a total of two independent experiments with at least three mice per group per experiment. <b>H-K</b>-Gene expression analyses showing mean± SD (copies) of IL-17A (in <b>H</b>), CCL20 (in <b>I</b>), matrix metalloproteinases (MMP)-3 (in <b>J</b>) and MMP-9 (in <b>K</b>) mRNA transcripts in cornea epithelia of nude mice that received CD4⁺T cells isolated from donor mice that had received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Data represents mean ± SD. Experiments were repeated two times with at least three mice per group per experiment.</p

Charcterization of intraepithelial lymphocytes (IELs) in the normal murine conjunctiva.

<p>Representative flow cytometry analysis of cells isolated from ocular surface (A) or spleen (B) stained with CD3 antibody and γδ (GDTCR), CD8α, NK1.1 and CD103 markers. (+) = positive cells, (–) =  negative cells. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”, circled population on far left panels), subsequently gated based on forward scatter height vs. forward scatter area (FSC-A) and propridium iodide live/dead exclusion (“live cells”, not shown). Numbers in the quadrants indicate the percentage of cells of one representative experiment. SSC-A = side scatter area.</p