Somatostatin Actions via Somatostatin Receptors on the Ocular Surface Are Modulated by Inflammatory...

Somatostatin Actions via Somatostatin Receptors onthe Ocular Surface Are Modulated by InflammatoryProcesses

Ivonne Minsel, Rolf Mentlein, Saadettin Sel, Yolanda Diebold, Lars Brauer,Eckhard Muhlbauer, and Friedrich P. Paulsen

Departments of Anatomy and Cell Biology (I.M., L.B., E.M., F.P.P.) and Ophthalmology (S.S.), Martin Luther Universityof Halle-Wittenberg, D-06097 Halle/Saale, Germany; Department of Anatomy (R.M.), Christian Albrecht University ofKiel, D-24098 Kiel, Germany; and Department of Applied Ophthalmobiology (Y.D.), Instituto Universitario deOftalmobiologia Aplicada, University of Valladolid, 47005 Valladolid, Spain

Recent investigations support the presence of human somatostatin (SS) in the excretory system ofthe human lacrimal gland. To get deeper insights into a possible role of SS at the ocular surface andin the lacrimal apparatus, we investigated the distribution pattern of SS and its receptors 1–5(SSTR1-5) by means of RT-PCR, real-time RT-PCR, Western blot and immunodot blot analysis as wellas immunohistochemistry in lacrimal gland, tear fluid, conjunctiva, cornea, nasolacrimal duct ep-ithelium, and conjunctival (HCjE) and corneal (HCE) epithelial cell lines. Cell culture experimentswith HCjE and HCE were performed to analyze a possible impact of SS and inflammatory mediatorson the regulation of SSTR. The results confirmed the presence of SS in lacrimal gland and tear fluid,whereas it was absent at the protein level in all other tissues and cell lines investigated. Expressionof SSTR1, -2, and -5 was detectable in lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts.HCjE expressed only hSSTR1 and -2, and HCE revealed only SSTR2. SSTR3 and -4 were not detectedin any of the analyzed samples or cell lines. In vitro on cultured immortalized HCjE cells SS leads toa concentration-dependent down-regulation of SSTR1 mRNA but does not affect SSTR2 mRNAexpression. Relative expression of SSTR1 and -2 is differentially modulated by proinflammatorycytokines and bacterial components, suggesting that the expression of both receptors is immu-nomodulated. Our data support an autocrine and paracrine role of SS in the lacrimal system andat the ocular surface and implicate a role of SS in corneal immunology. (Endocrinology 150:2254–2263, 2009)

Bacterial keratitis, conjunctivitis, and dry eye are among themost common problems presenting to the ophthalmologist.

Bacterial keratitis and conjunctivitis comprise an importantcomponent of numerous infections of the eye, particularly afterpenetrating corneal injuries and after long-term contact lenseswear and subsequent to refractive corneal surgery (1–4). Dry eyeis a multifactorial disease of the tears and ocular surface thatresults in symptoms of discomfort, visual disturbance, and tearfilm instability with potential damage to the ocular surface. It isaccompanied by increased osmolarity of the tear film and in-flammation of the ocular surface (5).

Studies on cultivated rat brain cells have revealed that thecyclic polypeptide somatostatin (SS) can be induced in these cells

by proinflammatory cytokines such as IL-1ß, TNF�, and IL-6 (6,7). Further studies have revealed that SS is induced and secretedby human fatty tissue (adipocytes) under inflammation and tis-sue infection and may influence the course of inflammation (8).

SS is the natural ligand of the somatostatin receptor. It isproduced in two variants consisting of 14 (ss-14) or 28 (ss-28)amino acids, both of which are derived from a common precur-sor molecule, preprosomatostatin, by posttranslational proteo-lytic processing (9, 10). SS is present in many tissues of the humanbody at discrete concentrations. Large amounts are excreted inthe gastrointestinal tract, pancreas, and peripheral and centralnervous systems. Lesser amounts have been demonstrated in thekidney, adrenal glands, lungs, thyroid, testicles, and spleen (11,

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2009 by The Endocrine Societydoi: 10.1210/en.2008-0577 Received April 22, 2008. Accepted December 12, 2008.First Published Online December 23, 2008

Abbreviations: CT, Cycle threshold; DNase, deoxyribonuclease; HCE, human corneal ep-ithelial; HCjE, human conjunctival epithelial; LPS, lipopolysaccharide; PGN, peptidoglycan;RNase, ribonuclease; SS, somatostatin; SSTR, SS receptor; TBS, Tris-buffered saline;

G R O W T H H O R M O N E - S O M A T O S T A T I N - G R H

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12). SS activity is correspondingly diverse, e.g. it inhibits therelease of the GH, somatotropin, by the anterior pituitary (13) orinhibits endocrine and exocrine processes in the pancreas andother organs (14). Beyond this, it functions as a neurotransmitterin the brain and peripheral nerve cells (15).

SS’s effects are mediated by five high-affinity, G protein-cou-pled surface receptors (sstr1-5). They are present in many tissuesand code the functional response to somatostatin bindingthrough their various distribution patterns and via the signaltransduction mechanisms coupled to them in a specific manner(15). The five types of somatostatin receptors can be differenti-ated into two subgroups on the basis of structural differences andpharmacological characteristics, the SRIF1 and SRIF2 receptors.The SRIF1 receptor group, consisting of SSTR2, -3, and -5, ischaracterized by its binding capacity for the classical somatosta-tin analogs (octreotide, lanreotide). The SRIF2 group, to whichSSTR1 and -4 belong, is characterized by a very minimal analogbinding capacity.

Various studies demonstrated the presence of SS on the ocularsurface in animals. In the late 1980s to early 1990s, it was dem-onstrated that SS is produced in the Harderian glands of rats,which seems to represent the equivalent of the human Meibo-mian glands (16, 17). SS has also been demonstrated in tear fluidof rabbits (18). Immunohistochemical studies communicated byLober (19) in a congress abstract support the production of SS inthe human lacrimal gland and at the same time raise the questionof the functional significance of this hormone at the ocular sur-face. It is as yet unknown whether SS is present in the human tearfilm and thus whether it may exert a possible influence as animmune modulator in corneal infections or inflammation in theabove mentioned clinical ocular surface disorders.

In the present study, we therefore reevaluated the immuno-histochemical finding of Lober (19) whether SS is produced bythe lacrimal gland and whether it is secreted into the tear fluid.Moreover, we characterized the SSTR equipment of lacrimalgland, cornea, conjunctiva, and nasolacrimal ducts and tested invitro possible immunomodulatory functions of somatostatin atthe ocular surface.

Materials and Methods

Tissues and cell linesThe study was conducted in compliance with institutional review

board regulations, informed consent regulations, and the provisions ofthe Declaration of Helsinki.

Lacrimal glands, upper eye lids (conjunctivas), corneas, and naso-lacrimal systems (consisting of lacrimal sac and nasolacrimal duct) wereobtained from cadavers (four males, 11 females, aged 53–89 yr) donatedto the Department of Anatomy and Cell Biology, Martin Luther Uni-versity Halle-Wittenberg, Germany. All tissues used were dissected fromthe cadavers within a time frame of 4 up to 12 h postmortem. The donorswere free of recent trauma, eye and nasal infections, or diseases involvingor affecting lacrimal function. Control tissues (hypophysis, pancreas, orlung) were obtained from the same cadavers.

After dissection, tissues from the right eye of each cadaver were pre-pared for paraffin embedding and fixed in 4% paraformaldehyde (in 13cases parts of the lacrimal gland were additionally frozen at �80 C forimmunohistochemistry on frozen sections); tissues from each left eye

were used for molecular biological investigations and were immediatelyfrozen at �80 C. After 1 wk, all tissues fixed in 4% formalin weredehydrated in graded concentrations of ethanol and embedded inparaffin.

Simian virus-40-transformed human corneal epithelial (HCE) cells (akind gift from Kaoru Araki-Sasaki, Tane Memorial Eye Hospital, Osaka,Japan) (20) and a human, spontaneously immortalized epithelial cell linefrom normal human conjunctiva (IOBA-NHC, here referred to as HCjEcells) (21) were cultured as monolayers and used for stimulationexperiments.

Collection of tear fluid and aqueous humorTear fluid (reflex tears) was collected from the lower conjunctival

sacs of six healthy human volunteers by Schirmer strips as previouslydescribed by others (22). Part of Schirmer strips were pressed out by handin a plastic bag to obtain pure tear fluid. Other Schirmer strips weretreated with NaCl/HEPES or acetonitrile/0.1% trifluoroacetic acid toextract SS. Thus, collected tear samples of each volunteer were pooled,immediately frozen, and stored at �20 C for use in experiments.

Cell cultureThe 5 � 106 HCE and HCjE cells were seeded into 25-cm2 flasks and

cultured in DMEM (DMEM F12; PAA Laboratories GmbH, Pasching,Austria) containing 10% fetal calf serum (Biochrom AG, Berlin,Germany). At confluency, the cells were exposed to different concentra-tions (0.1, 1.0, and 10.0 ng/ml) of synthetic SS-14 (Bachem, Bubendorf,Switzerland) for 6 h or IL-1� (10 ng/ml; ImmunoTools, Friesoythe, Ger-many), IL-1ß (10ng/ml; ImmunoTools), TNF� (10 ng/ml; Immuno-Tools), lipopolysaccharide (LPS; 1 �g/ml; Santa Cruz Biotechnology,Inc., Heidelberg, Germany), and peptidoglycan (PGN; 1 �g/ml; SantaCruz Biotechnology) for 6 and 24 h in serum-free DMEM with 0.05%BSA. Cells were used for RT-PCR and real-time RT-PCR analysis.

HCE and HCjE cells were also seeded on glass coverslides in six-wellculture plates and incubated. At confluence, cells were exposed to IL-1�(10 ng/ml), IL-1ß (10 ng/ml), TNF� (10 ng/ml), LPS (1 �g/ml), or PGN(1 �g/ml) in serum-free DMEM with 0.05% BSA for 24 h (all values aregiven as final concentration). Cytokine and bacterial toxin concentra-tions were used as published recently (23–25). All experimental proce-dures were performed under normoxic conditions (20% pO2, 5% CO2).At the end of each experiment, the cells on glass coverslides were fixed for5 min in 4% paraformaldehyde and processed for immunohistochemistry.

RNA preparation and cDNA synthesisFor conventional RT-PCR, tissue biopsies from 15 lacrimal glands,

conjunctivas, corneas, and nasolacrimal systems were crushed in an ag-ate mortar under liquid nitrogen and then homogenized in 5 ml peqgoldRNA pure solution (peqLab Biotechnologie, Erlangen, Germany) with aPolytron homogenizer. Insoluble material was removed by centrifuga-tion (12,000 � g, 5 min, 4 C). Total RNA was isolated using Rneasy kit(QIAGEN, Hilden, Germany). Crude RNA was purified with isopropa-nol and repeated ethanol precipitation, and contaminating DNA wasdestroyed by digestion with ribonuclease (RNase)-free deoxyribonucle-ase (DNase) I (30 min, 37 C; Boehringer, Mannheim, Germany). Con-tamination of the purified RNA by genomic DNA additionally was pre-vented by incubation with RQ1-RNase-free DNase (Promega GmbHMannheim, Germany). To proof DNA degradation, PCR with the spe-cific DNA-primers for SS, SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 aswell as ß-actin was performed. In no case was amplification obtainableusing the purified RNA as a template. The DNase was heat denatured for10 min at 65 C. Five hundred nanograms RNA were used for eachreaction: cDNA was generated with 50 ng/�l (20 pmol) oligo(deoxythy-midine)15 primer (Amersham Pharmacia Biotech, Uppsala, Sweden) and0.8 �l superscript RNase H� reverse transcriptase (100 U; Life Tech-nologies, Inc., Paisley, UK) for 60 min at 37 C. The ubiquitously ex-pressed ß-actin, which proved amplifiable in each case with the specificprimer pair, served as the internal control for the integrity of the trans-lated cDNA.

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PCRPCR amplification was carried out with 2 �l cDNA in a final volume

of 30 �l containing 0.18 �l Taq-polymerase, 0.9 �l 50 mM MgCl2, 3 �lPCR buffer, 0.6 �l deoxynucleotide triphosphate, and 0.6 �l primer mix,using the generated and designed primer combinations shown in Table1. After 3 min of heat denaturation at 94 C, the PCR cycle consisted of:1) 94 C for 30 sec; 2) 56 C [human SS (hSS)], 57 C (SSTR1, SSTR2), 59C (SSTR3, SSTR4), 61 C (SSTR5) for 30 sec each; and 3) 72 C for 45 sec.30 cycles were performed with each primer pair. The final elongationcycle consisted of 72 C for 10 min. The primers were synthesized byMWG-Biotech AG (Ebersberg, Germany). Ten microliters of the PCRwere loaded onto an agarose gel, and after electrophoresis the amplifiedproducts were visualized via fluorescence. Base pair values were com-pared with gene bank data. PCR products were also confirmed by BigDyesequencing (Applied Biosystems, Foster City, CA). To estimate theamount of amplified PCR product, we performed a ß-actin PCR withspecific primers for each investigated tissue. For this additional PCR, weused the above-mentioned conditions.

Real-time RT-PCR for hSSTR1 and hSSTR2Samples were analyzed by real-time RT-PCR using an Opticon 2

system (MJ Research, Waltham, MA). Reactions were performed usingSYBR green master mix (Applied Biosystems, Darmstadt, Germany) asa double-stranded DNA-specific fluorescent dye, with the appropriateprimer sets: SSTR1, forward, 5�-GGC GAA ATG CGT CCC AG-3� andreverse, 5�-CGG AGT AGA TCA AAG AGA TCA GGA-3�; hSSTR2,forward, 5�-GTC CTC TGC TTG GTC AAG GTG-3� and reverse, 5�-TGG TCT CAT TCA GCC GGG ATT-3�; 18S-rRNA, forward, 5�-ACTCAA CAC GGG AAA CCT CAC C-3� and reverse, 5�-CAA GAG ATGGCC ACG GCT GCT-3� (for SSTR1, SSTR2, and 18S-rRNA, respec-tively). 18S-rRNA was used because its expression was not influenced byany of the mediators under investigation. PCR was initiated with 2 minat 50 C, followed by 1.5 min at 95 C. The program continued with 30cycles of 20 sec at 95 C and 60 sec at 60 C. Each assay included duplicatesof each cDNA sample and a no-template control for SSTR1 and SSTR2.The parameter cycle threshold (CT) is defined as the cycle number atwhich fluorescence intensity exceeds a fixed threshold. Relative amounts

of mRNA expression for SSTR1, SSTR2, and 18S-rRNA were calculatedusing the ��CT method (26). The expression of 18S-rRNA was used tonormalize samples for the amount of cDNA used per reaction. To con-firm the amplification, the resulting real-time PCR products were ana-lyzed by dissociation curves, visualized in an agarose gel (SSTR1, 75 bp;SSTR2, 85 bp; 18S-rRNA, 111 bp), and sequenced via Big Dye termi-nation sequencing.

AntibodiesAntibodies included rabbit antihuman SS (Dako, Glostrup, Den-

mark; A0566); goat antihuman SSTR1 (Santa Cruz Biotechnology,Santa Cruz, CA; sc-11603); goat antihuman SSTR2 (Santa Cruz Bio-technology; sc-11606); goat antihuman SSTR3 (Santa Cruz Biotechnol-ogy; sc-11614); goat antihuman SSTR4 (Santa Cruz Biotechnology; sc-11619); and goat antihuman SSTR5 (Santa Cruz Biotechnology;sc-11623).

Western blot analysisFor Western blots, human tissue from lacrimal gland, conjunctiva,

cornea, and nasolacrimal ducts of cadavers (standardized ratio: 100 mgwet weight to 400 �m buffer containing 1% sodium dodecyl sulfate and4% 2-mercaptoethanol) was extracted as described in detail by Brauer etal. (27), and the protein content was measured with the Bradford dye-binding procedure (Bio-Rad, Hercules, CA). Proteins (20 �g) were re-solved by reducing 10% SDS-PAGE, electrophoretically transferred atroom temperature for 1 h at 0.8 mA/cm2 onto 0.1 �m pore size nitro-cellulose membranes, and fixed with 0.2% glutaraldehyde in PBS for 30min. Protein bands were detected with primary antibodies to SSTR1(goat polyclonal, 1:4,000), SSTR2 (goat polyclonal, 1:4,000), andSSTR5 (goat polyclonal, 1:4,000), and secondary antibodies (antigoatIgG conjugated to horseradish peroxidase, 1:10,000; Dako; applyingchemiluminescence (ECL-Plus; Amersham Pharmacia, Uppsala, Swe-den). Lung tissue was used as positive control. Specificity of the primaryantibodies was tested by competition with the corresponding syntheticpeptide: sc11603P for SSTR1, sc11606P for SSTR2, and sc11623P forSSTR5 (all from Santa Cruz Biotechnology).

ImmunohistochemistryLacrimal glands were cryosectioned or sectioned and dewaxed. Im-

munohistochemical staining was performed with antibodies to SS (1:25),SSTR1 (1:30), SSTR2 (1:30), SSTR3 (1:30), SSTR4 (1:30), and SSTR5(1:30). Cryosections were pretreated with testicular hyaluronidase(Boehringer) in Tris-buffered saline (TBS) in a moist chamber at 37 C for30 min. The sections were washed three times with TBS and incubatedwith goat serum for 45 min at room temperature. Incubation with theprimary antibody was carried out for 60 min at room temperature. De-waxed sections were microwaved for 10 min and nonspecific bindinginhibited by incubation with porcine normal serum (Dako) 1:5 in TBS.The primary antibodies were applied overnight at room temperature.The secondary antibody porcine antirabbit (for both cryosections anddewaxed sections) or porcine antigoat was incubated at room temper-ature for at least 4 h. Visualization was achieved with peroxidase-labeledstreptavidin-biotin and diaminobenzidine for at least 5 min. After coun-terstaining with hemalum, the sections were mounted in Aquatex (Boehr-inger). Two negative control sections were used in each case: one wasincubated with the secondary antibody only, the other with the primaryantibody only. Sections of human hypophysis or pancreas were used forpositive control. Furthermore, specificity of the primary antibodies forSSTR was tested by competition with the corresponding synthetic pep-tide, which are blocking peptides sc11603P for SSTR1, sc11606P forSSTR2, sc11614P for SSTR3, sc11619P for SSTR4, and sc11623P forSSTR5 (all from Santa Cruz Biotechnology). Specificity of the primaryantibody for SS was evidenced by preincubation of the anti-SS antibodywith synthetic SS-14 (Bachem; H-1490) for 1 h. The slides were examinedwith a Axiophot microscope (Zeiss, Jena, Germany).

TABLE 1. Primers used for RT-PCR

Name Primer (5�–3�)Size(bp)

SS Sense, GAT GCT GTC CTG CCG CCT CCA G 349Antisense, ACA GGA TGT GAA AGT CTT CCA-

SSTR1 Sense, GGA GCC GGT TGA CTA TTA CGC C 107Antisense, AGG TGC CAT TAC GGA AGA CGC

SSTR1 Sense, GGA ACT CTA TGG TCA TCT A 541Antisense, GAG GGC CAC CAT GCG CAT CTT

SSTR2 Sense, CCG CTA TGC CAA GAT GAA GAC C 182Antisense, TGC GGG TGA ACT GAT TGA TGC C

SSTR2 Sense, CGG AGC AAC CAG TGG GGG A 376Antisense, GGG TTG GCA CAG CTG TTA GC

SSTR3 Sense, TTA TGG CTT CCT CTC CTA CCG C 120Antisense, TCC TCC TCC TCA GTC TTC TCC G

SSTR3 Sense, CCC GCG GCA TGA GCA CCT G 376Antisense, GGG TTG GCA CAG CTG TTG G

SSTR4 Sense, CCT GTG CTA CCT GCT CAT CGT G 137Antisense, CAT CCA GCA GAG CAC AAA GAC G

SSTR4 Sense, GCA GAC ACC AGA CCG GCT C 371Antisense, GGG TTG GCG CAG CTG TTG G

SSTR5 Sense, CCA AGA TGA AGA CCG TCA CCA A 181Antisense, CAG AAG ACA CTG GTG AAC TGG TTG

SSTR5 Sense, AGG AGG GCG GTA CCT GCA A 361Antisense, GGG TTG GCA CAG CTG TTG G

ß-Actin Sense, AAG AGA TGG CCA CGG CTG CT 275Antisense, TCC TTC TGC ATC CTG TCG GCA

2256 Minsel et al. Somatostatin at the Ocular Surface Endocrinology, May 2009, 150(5):2254–2263

Moreover, immunohistochemistry was performed on stimulated andnonstimulated HCE and HCjE cells on glass coverslides as describedabove.

Immunodot blotAn immunodot blot assay was performed for human SS protein de-

tection in tears. Collected tear fluid samples were centrifuged to removeany cell debris. Standard curves were generated with synthetic SS-14(Bachem). The peptide was diluted serially. Three microliters of eachsample (three samples from each volunteer including pure tear fluid,NaCl/HEPES extracted tear fluid, and acetonitrile/0.1% trifluoroaceticacid extracted tear fluid) were spotted in triplicate on nitrocellulosemembrane. The membrane was blocked with 10% nonfat dry milk andTBS-0.1% Tween 20 at room temperature for 1 h and then incubated at4 C overnight with rabbit antihuman SS (diluted 1:50). The membraneswere then incubated at room temperature for 1 h with goat antirabbitIgG-horseradish peroxidase-labeled (Santa Cruz Biotechnology; sc2004,diluted 1:100,000 in blocking solution). After washing in TBS-0.1%Tween 20, the membranes were developed and visualized using the ECLchemiluminescence system (Amersham Biosciences, Freiburg, Germany)followed by exposure to autoradiographic X-Omat AR film (EastmanKodak, Rochester, NY). For quantification of SS, the signal intensities ofaliquots were compared directly with those of simultaneously preparedstandards consisting of known amounts of SS, using PC BAS densitom-etry software (LAS-1000; Fujifilm Medical Systems, Stamford, CT). Be-cause different amounts of tear fluid show different adsorption the dotblot is taken as a semiquantitative experiment.

Statistical analysisData are expressed as mean � SEM. Statistical significance was de-

termined by the Wilcoxon (Student’s t test) signed-rank test and Mann-Whitney U test. P � 0.05 was considered statistically significant.

Results

SS is expressed in the human lacrimal glandSS-specific cDNA amplification products (349 bp) were de-

tected in all lacrimal glands, conjunctivas, and nasolacrimalducts (n � 15 for each tissue; Fig. 1 and Table 2) but were absentin all corneas analyzed (not shown). SS expression was immu-nohistochemically visible in all 15 lacrimal glands investigated(Fig. 1) but was not detectable in corneas, conjunctivas, andnasolacrimal ducts (not shown). In lacrimal gland SS is producedby some but not all acinus cells as well as cells of the excretoryduct system (Fig. 1). Both cryosections and dewaxed sectionsrevealed positive results (Fig. 1).

Tear fluid contains SSImmunodot blot assaying of human tear fluid revealed pres-

ence of SS. Densitometric analysis of differentially extracted tearfluid samples indicated a SS concentration varying between 0.07and 1.0 nmol/liter (Fig. 2). This concentration range was de-tected in tear fluid samples of all volunteers with individual vari-ation in between this range.

SSTR1, -2, and -5 are expressed in tissues of the lacrimalapparatus and ocular surface

SSTR1 and -2-specific cDNA amplification products (541and 376 bp, respectively) were detected in all lacrimal glands,corneas, conjunctivas, and nasolacrimal ducts (n � 8 for each

tissue; Table 2). SSTR3 and -4-specific cDNA amplificationproducts (213 and 1023 bp) were absent in all samples (Table 2).SSTR5-specific cDNA amplification products (360 bp) weredetected only in lacrimal glands, conjunctivae, and nasolac-rimal ducts but were absent in cornea (Table 2). The ß-actincontrol PCR was positive and of similar amount for all in-vestigated tissues as expected. Base pair values were equiva-lent to the expected DNA products in comparison with genebank data.

Samples from lacrimal glands, corneae, conjunctivae, and na-solacrimal ducts were dissected from eyes of cadavers (n � 7 foreach tissue), and extracts were tested for SSTR by Western blotanalysis (Fig. 3). Varying amounts of SSTR1 (at 62 kDa) andSSTR2 (at 62 kDa) were detected in all samples. Lacrimal glands,conjunctivae, and nasolacrimal ducts also contained varyingamounts of SSTR5 (at 65 kDa), whereas cornea did not comprise

FIG. 1. A, Ethidium bromide-stained agarose gel for visualization of RT-PCRamplification products derived from the following tissues (n � 15 for eachtissue): nasolacrimal duct (nd), lacrimal gland (lg), conjunctiva (con). PCRamplification products of human pancreas (pc) were used for positive control;blank lane (W) indicates the negative control without template DNA. Inaccordance with the DNA marker (M) and control, the distinct bands are visibleat about 349 bp for human somatostatin (SS). B–E, Immunohistochemicaldetection of somatostatin in lacrimal gland. B, C, and E, Paraffin-embeddedsections. D, Frozen section. In B, C, and E, somatostatin is visible as red staining.Arrow in C marks excretory duct; arrows in D and E mark positive reactions, E,positive control-somatostatin-positive cells in a pancreatic island. Insets revealhigher magnifications of a distinct area. Scale bar (B and E), 43 �m; (C and D)27 �m.

TABLE 2. Detection of SS and sstr1-5 by RT-PCR

Lacrimalgland Cornea Conjunctiva

Nasolacrimalduct HCE HCjE

SS � � � � Ø ØSSTR1 � � � � Ø �SSTR2 � � � � � �SSTR3 Ø Ø Ø Ø Ø ØSSTR4 Ø Ø Ø Ø Ø ØSSTR5 � Ø � � Ø Ø

�, Positive; Ø, negative.


any SSTR5. The staining was highly specific for each antibodyand was inhibited with the corresponding synthetic peptide(not shown). SSTR3 and -4 were not tested because RT-PCRrevealed no cDNA amplification products for the respectivereceptors.

Immunohistochemical analysis of lacrimal gland sections re-vealed positive reactivity for SSTR1, -2 and -5 in acinus cells oflacrimal glands (Fig. 3). Also here, the staining was highly spe-cific for each antibody and was inhibited with the correspondingsynthetic peptide (not shown).

Cell lines HCE and HCjE do not express SSSS-specific cDNA amplification products (349 bp) were not

detected in both cell lines HCE and HCjE (Table 2). Humanpancreas was used as positive control revealing a SS-specificcDNA amplification product at 349 bp (not shown). ß-Actinserved as internal control (not shown).

Detection of SSTR1 and -2-specific amplificationproducts in cell lines

SSTR1- and SSTR2-specific amplification products (541 and376 bp, respectively) were detected in the HCjE (Fig. 4 and Table2), whereas HCE yielded only 376 bp amplification productsindicating expression of SSTR2 but absence of SSTR1 (Fig. 4 andTable 2). All other receptors SSTR3, -4, and -5 could not bedetected in any of the two cell lines (Fig. 4 and Table 2). Humanpancreas was used as positive control, revealing SSTR-specificcDNA amplification products at 541 bp (SSTR1), 276 bp(SSTR2), 213 bp (SSTR3), 1023 bp (SSTR4), and 360 bp(SSTR5) (Fig. 4). ß-Actin served as internal control (Fig. 4).

Stimulation of HCjE with recombinant SSTo test whether SS has an influence on SSTR1 and -2 regu-

lation of ocular surface epithelial cells, HCjE cells were culturedand treated with different concentrations (0.1, 1.0, and 10.0ng/ml) of SS for 6 h. Afterward real-time RT-PCR was per-formed. The results reveal a significant concentration-dependentreduction of the relative SSTR1 expression after 1.0 and 10.0ng/ml SS treatment, whereas 0.1 ng/ml SS showed no effect (Fig.5). The relative SSTR2 expression was not significantly changedby all different SS concentrations applied (Fig. 5).

Regulation of SSTR1 and -2 in HCjE and HCE byinflammatory cytokines and bacterial components

To investigate the regulation of SSTR1 and -2 by inflamma-tory cytokines and bacterial components and products, we an-

FIG. 2. Dot-blot analysis for SS of human tear fluid. Tear fluid (tf) was collectedby Schirmer strips from a volunteer. After detection of SS with an anti-SSantibody, optical densitometry against standards was performed and the valueswere plotted on a histogram. Because different amounts of tear fluid showdifferent adsorption, the dot blot is taken as a semiquantitative experiment. A,Extraction of tf with NaCl/HEPES. B, Extraction of tf with acetonitrile/0.1%trifluoroacetic acid. C, Pure tear fluid.

FIG. 3. A, Western blot analysis of somatostatin receptors 1, -2, and -5 in lung(lu), hypophysis (hy), lacrimal gland (lg), nasolacrimal duct (nd), conjunctiva (con),and cornea (cor). B–G, Immunohistochemical detection of human SSTR1 (B),SSTR2 (D), and SSTR5 (F) on acinar cells (arrows) of lacrimal gland visible by a redreaction product. Tissue specimen from human hypophysis served as positivecontrol (C, E, and G). Insets reveal higher magnifications of a distinct area. Scalebar (B, C, D, and F) 43 �m; (E and G) 27 �m.


alyzed the influence of IL-1�, IL-1ß, TNF�, LPS, and PGN onmonolayers of HCjE and HCE.

HCjEReal-time RT-PCR revealed induction of SSTR1 mRNA after

stimulation with 10 ng/ml IL-1�. Six hours after exposure toIL-1�, basal SSTR1 mRNA levels increased nearly 2-fold com-pared with the unstimulated samples (Fig. 6). In contrast, TNF�

(10 ng/ml) significantly down-regulated basal SSTR1 levels tonearly 50% (Fig. 6). No significant change was obvious aftertreatment with IL-1ß, LPS, or PGN (Fig. 6). Twenty-four hoursafter exposure, no significant change was found after treatmentwith none of the stimulants (Fig. 6). SSTR2 mRNA was inducedafter stimulation with 10 ng/ml IL-1� or IL-1ß, or 1 �g/ml LPSor PGN. Six hours after exposure to IL-1�, IL-1ß, LPS, and PGN,basal SSTR2 mRNA levels increased nearly 1.8-, 2.2-, 2.7-, and2.4-fold, respectively (Fig. 6). No significant change was obviousafter treatment with TNF�. After twenty-four hours, SSTR2mRNA levels of IL-1ß, LPS, and PGN decreased back to basallevels (Fig. 6). IL-1� down-regulated SSTR2 to nearly 40% inconjunctiva (Fig. 6).

HCEReal-time PCR revealed induction of SSTR2 mRNA after 6 h

stimulation with 10 ng/ml IL-1�, IL-1ß, or 1 �g/ml PGN. SSTR2mRNA levels increased 2.8-, 1.4-, and 1.5-fold, respectively,whereas TNF� and LPS revealed no significant effect (Fig. 6).After 24 h SSTR2 mRNA levels were significantly up-regulatedby IL-1ß (1.6-fold), LPS (1.7-fold), and PGN (1.5-fold). IL-1�

led to a decrease in control levels and TNF� also revealed nochange at this time point.

HCE and HCjE cells express SSTR1 and -2Positive immunoreactivity was visible in all independently

cultured epithelial cells of the two cell lines analyzed before stim-ulation (Fig. 7, untreated). After stimulation with the differentcytokines and bacterial components, cultured epithelial cells re-vealed clear positive staining for SSTR1 and SSTR2 (Fig. 7, IL1�

or PGN). Cells showed expression of both SSTR1 and SSTR2,

regardless of whether they were stimulated. Subjectively the im-munohistochemical reaction appeared stronger in stimulatedcells that revealed also induction of SSTR1 and -2 in real-timePCR experiments than in untreated cells (IL-1� in HCE andHCjE; PGN and IL-1ß in HCE and HCjE, only for SSTR2; andLPS only in HCjE for SSTR2; Fig. 7).

Discussion

The results of the present study suggest that SS is produced by theacinar cells of the lacrimal glands and secreted into the tear fluid.SS-specific cDNA amplification products are demonstrable inconjunctiva and samples of nasolacrimal ducts but are absent incornea as well as corneal and conjunctival epithelial cell lines. Atthe protein level except for lacrimal gland, SS is not demonstrablein epithelia of the human ocular surface neither in cornea, con-junctiva, corneal, and conjunctival epithelial cell lines nor innasolacrimal ducts. SSTR1, -2 and -5 can be demonstrated at the

FIG. 4. Ethidium bromide-stained agarose gels for visualization of PCRamplification products derived from HCE and HCjE cell lines. In accordance withthe DNA marker (Bp), the distinct DNA bands are visible at 376 bp for humanSSTR2 in HCE and HCjE cells and at 541 bp for human SSTR1 in HCjE cells. APCR amplification product of human pancreas (C) was used for positive controlfor all SSTR. ß-Actin PCR control is shown additionally at 178 bp. P, Appliedsample.

FIG. 5. Real-time RT-PCR of cultured spontaneously immortalized conjunctivalepithelial cells (HCjE). Cells were stimulated for 6 h with different concentrationsof recombinant somatostatin. The results indicate that the relative expression ofSSTR1 is significantly down-regulated in a concentration-dependent manner (A),whereas the relative expression of SSTR2 is not influenced by somatostatin attime point 6 h (B). The fold increase in transcript levels over or under controls isexpressed as mean � SEM, and statistical significance (P � 0.05) is indicated by anasterisk.


mRNA and protein levels in lacrimal gland, conjunctiva, cornea,and nasolacrimal ducts. This is in contrast to findings by Klisovicet al. (28), who found neither SSTR1 nor SSTR2 in human cor-neal epithelium. Moreover, our present results indicate thatHCjE and HCE cell lines both express SSTR2 and HCjE in ad-dition SSTR1, whereas SSTR3-5 are absent in both cell lines.These findings suggest an interference of autocrine as well asparacrine effects of SS on the ocular surface epithelia and struc-tures of the lacrimal apparatus.

Based on known antisecretory effects of SS, inhibition of fluidsecretion in the lacrimal glands could be presumed. Bausher andHorio (29) already described a modulating influence of SS on theproduction of aqueous humor in the rabbit. A similar effect hasbeen pointed out by Kapicioglu et al. (30) after ip injection ofoctreotide, a somatostatin analog, in the rat. Furthermore, theantiproliferative characteristics of SS lead to a consideration ofits possible functional importance in maintaining the transpar-ency of the cornea. This has been supported by immunohisto-chemical observations, which have demonstrated that the hu-man cornea exhibits SSTR1 and -2 in the stroma and on theendothelial cells (28). The immunomodulatory effects mediatedby SS, such as their influence on lymphocyte proliferation or ILand cytokine release (31), could also lead to consideration of an

effect on the ocular surface in corneal infec-tions and inflammatory processes.

The described activities of SS are medi-ated on the ocular surface mainly via SSTR1and -2. On the basis of expression analyzesof somatostatin receptors, it could be dem-onstrated that SSTR1 and -2 are the mostfrequently expressed receptors in human oc-ular tissues (28). These findings correlatewith distributions of somatostatin receptorsfound in other studies examining tissue andcell lines (32). SSTR5 is only sporadicallydemonstrable on the ocular surface, andSSTR3 and -4 were not detected in any of thespecimens examined. Interestingly, it couldbe shown on the example of the human con-junctival epithelial cell line that SSTR1mRNA is significantly down-regulated afterconcentration-dependent SS stimulation(1 and 10 ng/ml). In contrast, hSSTR2 isindependent of SS regulation in the chosenconcentration range. This finding is ratherunexpected because down-regulation and/ora desensitization were to be expected in thepresence of SS. It may be assumed that thisregulatory mechanism occurs only after alengthy period of stimulation. This is possiblyassociated with the functional importance ofSSTR2 and the maintenance of certain regu-latory mechanisms.

Neuropeptide hormones are involved inthe complex regulatory processes betweenthe human neuroendocrine system and theimmune system (33). An involvement of SS

and its receptors is also assumed in relation to these interactions.Numerous studies have been performed to examine the distri-bution of SS and somatostatin receptors in endocrine tissue (12).However, the complex role of SS in the human immune systemhas not been well elucidated to date. Up to the present, it couldbe shown that various neuropeptides, including SS are capable ofinducing cytokine secretion by T cells. Beyond this, SS-inducedinhibition of secretory activity of IL-8 and IL-1�, which had beentriggered by TNF� and bacteria, has been described in two in-testinal cell lines (33). The SSTR2 is expressed by monocytes,macrophages, and dendritic cells in the human organism and canbe significantly up-regulated through stimulation by LPS (34).The role of SS in immunomodulatory tissues has previously beenstudied in animal models. Thus, for example, it could be dem-onstrated that SS acts directly on the T cell somatostatin receptor(SSTR2), consequently down-regulating interferon-� synthesis(32, 35). In rats, SS is also synthesized and secreted by T and Blymphocytes of the thymus and spleen (36).

To date, no findings are available to answer the question as towhether SS is important as an immune modulator in cornealinfections and inflammation of the ocular surface. Thus, the goalof our studies has been to analyze the influence of cytokines(TNF�, IL-1�, IL-1�) and bacterial components (PGN, LPS) on

FIG. 6. Real-time RT-PCR of cultured conjunctival (HCjE) and corneal epithelial (HCE) cell lines. Cells werestimulated for 6 (left side) or 24 h (right side) with LPS, PGN, TNF�, IL-1�, or IL-1�. The results indicate thatonly IL-1� significantly (*) up-regulates SSTR1 expression after 6 h in conjunctival epithelial cells, whereasTNF� down-regulates the receptor. In contrast SSTR2 is up-regulated by bacterial components and ILs inboth HCjE and HCE after 6 h. In conjunctiva this effect is lost after 24 h, whereas in cornea, PGN and IL-1ßlead to further up-regulation. SSTR2 is up-regulated with LPS first after 24 h in cornea, whereas inconjunctiva it is up-regulated already after 6 h. The fold increase in transcript levels over or under controlsis expressed as mean � SEM, and statistical significance (P � 0.05) is indicated by an asterisk.


somatostatin receptor expression in human corneal and conjunc-tival epithelial cell lines, respectively, after periods of 6 and 24 h.

The relative expression analyses are limited to SSTR1 and -2as the other receptor types, SSTR3, -4 and -5, are not present onHCjE and HCE cell lines. The ubiquitously demonstrable SSTR2could be studied in both cell lines, whereas, in contrast, SSTR1is expressed only by HCjE cells.

Studies have shown that bacterial components, LPS and PGN,as well as the proinflammatory cytokine IL-1� influence themRNA of SSTR2 only, underlining the fact that they do notregulate SSTR1 at any time examined. The stimulants lead to asignificant relative increase in mRNA for SSTR2 in HCjE cellsafter 6 h of stimulation. This effect is reversed after 24 h. In HCEonly PGN and IL-1� lead to an up-regulation of SSTR2 after 6 h.After 24 h of stimulation, the up-regulation of SSTR2 is stillsignificant in comparison with HCjE. The regulation of SSTR2by LPS, PGN, and IL-1� is thus of a longer duration in HCE thanHCjE cells. The proinflammatory cytokine TNF� exclusivelyregulates SSTR1 in HCjE. After 6 h, TNF� leads to a significantreduction in the relative mRNA expression, which is then re-versed after 24 h. These findings correlate with studies per-formed by Yan et al. (37), who also demonstrated a reduction inmRNA expression of SS and SSTR1, -2, and -5 after TNF�-stimulation in human coronary endothelial cells. The lack ofTNF� influence on SSTR2 in HCjE may be due to organ-specificactivity.

Gene expression levels are apparently very sensitive to IL-1�

stimulation, because, after a period of 6 h, IL-1� exposure con-sistently leads to a significant increase in SSTR1 and -2 receptorexpressions in the individual epithelial cell lines. This relativeincrease in expression is no longer demonstrable after a period of24 h, so that it can be assumed that receptor regulation by thiscytokine, but also by other stimulants, takes place at the mRNA

level and reaches its peak after 6 h, and, after 24 h, has then beendown-regulated or inhibited.

Cultured immortalized HCE and HCjE cells revealed consti-tutive production of SSTR1 (HCjE only) and SSTR2 before stim-ulation. After 24 h stimulation with different cytokines and bac-terial components, the staining intensity of the cells increasedsubjectively. However, we did not quantify the staining intensityor the concentration of either protein. These will be interestingto determine in future investigations.

HCE and HCjE cells express SS mRNA but do not produce SSon the protein level in the unstimulated state. Further experi-ments will be interesting to find out whether distinct stimulantsare able to induce this SS expression.

The findings of expression analyses make it clear that cyto-kines and bacterial components on the ocular surface play animportant role in immunomodulatory processes. The stimulantsclearly influence the regulation of SSTR1 and -2 expressionsalthough a direct effect on somatostatin expression on the corneaor conjunctiva is improbable. The immunological properties ofsomatostatin in infections or inflammation may be mediated bychanges in the expression of receptors and thereby an increase inbinding capacity of SS. Beyond this, there may be a feedbackmechanism to the lacrimal gland, via which, for example, SSsecretion is up regulated by inflammatory mediators. For themost part, SS has an inhibitory effect on the secretory activity ofimmune cells as well as the immunoglobulin production of ac-tivated B lymphocytes, the cytokine production of activated Tlymphocytes, or the release of inflammatory cytokines by ba-sophils or monocytes (38, 39). Through this reduction in medi-ator secretion, the expression of SSTRs is also changed and theimmune response is thus regulated. Furthermore, SS changes theactivity of natural killer cells and the phagocytotic activity ofhuman monocytes and macrophages as well as chemotaxis ofneutrophils (39–42). In the human organism, the SSTR2 is ex-pressed by monocytes, macrophages, and dendritic cells; thus, SScan also act directly on immune cells and influence their func-tioning (34). Another possibility for the SS system to exert in-fluence via the paracrine pathway, as well as on the character-istics of immune cells via the autocrine pathway, would be thesecretion of SS or a neuropeptide similar to SS by the immunecells themselves. Dalm et al. (34) described the expression ofcortistatin but the lack of the presence of SS in monocytes, mac-rophages, and dendritic cells. Cortistatin is a peptide similar toSS and exhibits a high affinity for all five SSTRs (43). On the basisof the lack of demonstration of SS in their studies, the authorsaccord cortistatin a great importance in the mediation of immuneprocesses. Cortistatin-like activity could also occur on the ocularsurface. The lack of SS demonstration in ocular surface epithelia,however, does not necessarily imply that SS is replaced by cor-tistatin. Rather, a complementary mechanism between the twopeptides can be assumed, but this must be verified by furtherstudies.

Hypothetically the SS system at the ocular surface may havein this context a function in the open eye and the closed eyeenvironments (for review see Ref. 44). Different from the open-eye condition in closed-eye condition, there is a marked decreasein the rate of reflex-type tear secretion accompanied by a shift in

FIG. 7. Immunohistochemistry of SSTR1 and -2 in cultured HCjE and HCE cells.Red staining indicates expression of SSTR1 and -2 on epithelial cells. Images arerepresentative of a series of stimulated and untreated incubated cells. Scale bar,15 �m.


the lacrimal secretion from a mixed basal type secretion to anearly pure constitutive type secretion consisting almost entirelyof secretory IgA and a small amount of free secretory component.This results in the recovery of a tear sample after overnight eyeclosure in which sIgA consists of upwards of about 80% of thetotal protein. This is accompanied besides other effects by therecruitment within 2–3 h of eye closure of thousands of PMNcells and other entities into the preocular tear film (for review seeRef. 44). These changes are indicative of a subclinical state ofinflammation. It has been proposed this is suggestive of a fun-damental shift in the host-defense strategy from a passive-barrierdefense to an active inflammatory, complement, immune, andphagocyte-mediated process. It has been further suggested thatthis shift is necessitated to protect the vulnerable ocular surfacesfrom invasion by entrapped-proliferating microorganisms (45).An immunomodulatory function of SS may be possible in thiscontext for example by inhibiting excessive inflammation duringeye closure.

For the treatment of bacterial keratitis and conjunctivitis, SSmay hypothetically achieve clinical relevance due to its manifoldeffects and interactions with the immune system. As dry eye isalso associated with an increased rate of infection of the super-ficial ocular surface epithelium (5), and SS is present in tear fluid,its importance in dry eye may also be possible. SS can thus playan important role as an immunomodulator in inflammatory pro-cesses. On the other hand, it is possible that SS influences theformation of local androgens as has already been demonstratedfor various other hormones (for review see Ref. 46). In recentyears, circulating and locally produced androgens have comeinto the center of interest in relation to the pathogenesis of dryeye. A reduction in androgen levels below a specific limiting valueleads to an outpouring of proinflammatory cytokines, particu-larly IL-1ß, IL-2, interferon-� ,and TNF�, by circulating lym-phocytes in the glandular tissue (lacrimal glands, accessorylarimal glands, Meibomian glands) (47). However, this hy-pothetical function of SS at the ocular surface needs furtherelucidation.

Beyond this, the assumption could be made that somatostatinalso possesses immunological activity in the nasolacrimal ductsas SSTR1, -2, and -5 are also demonstrable in this tissue. Theequipment of the nasolacrimal ducts with various nonspecificdefense systems which, for example, act against the developmentof dacryocystitis, has been described by Paulsen and colleagues(48–50) and Brauer et al. (27). Comparative anatomical studieshave furthermore discussed the possibility of re-resorption ofcomponents of tear fluid, which could represent a feedback sig-nal for the lacrimal gland (51). Although, the findings of thesestudies were obtained on the rabbit, they can be transferred to thehuman with only minimal limitations as the nasolacrimal ductsof the rabbit is very similar to that of humans. Thus, hypothet-ically the possible absorption of SS out of the tear fluid by the tearduct epithelium could represent an antisecretory feedback signalfor the lacrimal gland.

Taken together, we have shown that SS is secreted into thetear film and is able to act via SSTR1, -2, and -5 at the ocularsurface epithelia and tissues of the lacrimal apparatus (lacrimalgland, nasolacrimal ducts). In vitro at cultured immortalized

HCjE and HCE cell lines, SS leads to a concentration-dependentdown-regulation of SSTR1 mRNA but does not affect SSTR2mRNA expression in the investigated time range. Expression ofboth receptors is differentially modulated by proinflammatorycytokines and bacterial components. The results suggest an im-munomodulatory role for the SS system at the ocular surface andlacrimal apparatus in the healthy state (open and closed eye;corneal transparency) as well as under inflammatory conditions.Potential further modulating effects such as angio- or antiangio-genesis or wound healing need further elucidation.

Acknowledgments

We thank Martina Burmester, Ute Beyer, Michaela Risch, Susann Mos-chter, Stephanie Beileke, and Amalia Enriquez de Salamanca for excel-lent technical assistance.

Address all correspondence and requests for reprints to: Dr. FriedrichPaulsen, Department of Anatomy and Cell Biology, Martin Luther Uni-versity Halle-Wittenberg, Faculty of Medicine, Grosse Steinstrasse 52,D-06097 Halle (Saale), Germany. E-mail: [email protected].

This work was supported by Program Grants PA 738/6-1 and PA 738/1-5fromtheDeutscheForschungsgemeinschaft; theBundesministeriumfur,Bildung und Forschung, Wilhelm Roux Program, Program Grants FKZ09/17 (Halle, Germany); and Sicca Forschungsforderung of the Associationof German Ophthalmologists.

Disclosure Summary: The authors have nothing to disclose.

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