Presence and Localization of Neurotrophins and Neurotrophin Receptors in Rat Lacrimal Gland

Abstract

purpose. To determine which of the neurotrophins (NTs)—nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)—and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland.

methods. RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs.

results. RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes.

conclusions. Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.

Neurotrophins (NTs) are a family of growth factors first identified because of their ability to support differentiation and survival of the central and peripheral nervous systems. ^{1 2 3} This family consists of many well-studied factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) and -4 (NT4). In mammals, NTs act as growth factors for the development of the nervous system and the maintenance of the neural cell phenotype. Furthermore, the identification of equivalent or similar neurotrophins and their receptors in the vertebrates and invertebrates, suggest that they are highly conserved during evolution. NTs operate by activating multiple signaling pathways, including those that regulate physiological homeostasis and behavior. ^{4 5 6} The mechanism of action of the NTs was addressed by observing the effects of NGF on sympathetic neurons. NGF, similar to all the NTs, acts consistent with its retrograde transport along the axon from the synaptic terminal to the neuronal soma and also with a paracrine and autocrine mechanism. ^{7 8}  

Three main full-length neurotrophin receptors (NTrs), belonging to the tyrosine kinase protein (Trk) family, have been identified as high-affinity signal-transducing receptors for NTs. These receptors include TrkA (the first receptor to be discovered ⁹ ), TrkB, and TrkC. The p75 receptor is a member of the tumor necrosis factor family, but is missing an intracellular signal-transduction domain. When present with the other NTrs, it increases the affinity of NTs for their Trk receptors, although its function is still unclear (for a review see Ref. ¹⁰ ). The NTrs show high-binding affinity for the following growth factors: TrkA for NGF, TrkB for both BDNF and NT4, and TrkC for NT3. ^{11 12} All the NTs are able to bind p75. ^{12 13}  

It is well established that NTs interact with their receptors as functional homodimers and that after binding, each class of NTrs undergoes ligand-induced dimerization. ^{14 15 16} In addition, the NTrs show variable expression and location, which depends on their degree of glycosylation. This glycosylation is species specific. ¹⁷  

Recent evidence has demonstrated that secretion of NTs and expression of NTrs is not limited to neuronal cells. ^{18 19 20} Other tissues have also been shown to secrete NTs and express NTrs. NTs and NTrs play a key role in the development of nonneuronal tissues. ^{21 22} NTs and NTrs have been identified in the conjunctiva, not only under normal conditions, but also during the inflammation stage of the allergic reaction or after anterior segment injury. ^{23 24 25} NTs are also involved in clonal growth and differentiation of the corneal epithelium. ²⁶ NGF has been identified in the tears, ²⁷ suggesting a potential role of the lacrimal gland in this production. 

The objective of this study was to determine which NTs and NTrs are present in the adult rat lacrimal gland and their locations. We show that the NTs (NGF, BDNF, NT3, and NT4) and their NTrs (TrkA, TrkB, TrkC, and p75) are present in the lacrimal gland. NGF, BDNF, NT3, and NT4 are distributed throughout different types of lacrimal gland cells, whereas TrkA, TrkB, and TrkC are located on myoepithelial cell and ductal cell membranes. p75 was found exclusively in the myoepithelial cell membranes. 

Materials and Methods

Animals and Tissue Dissection

Use of animals conformed to the guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Schepens Eye Research Institute Animal Care and Use Committee. Male Sprague-Dawley rats weighing between 250 and 300 g were used in this study and were obtained from Taconic Farms (Germantown, NY). Animals were anesthetized for 1 minute in carbon dioxide, decapitated, and the lacrimal glands dissected. 

Materials

Rabbit polyclonal antibodies to the following NTs and NTrs and their corresponding peptides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and were used for both immunohistochemistry and Western blot analysis: NGF (sc-548), BDNF (sc-546), NT3 (sc-547), NT4 (sc-545), TrkA (sc-118), TrkB (sc-12), TrkC (sc-117), and p75 (sc-8317). Reverse transcription system and the PCR system (PCR Core System II) were purchased from Promega (Madison, WI). The fluorescein isothiocyanate (FITC)-conjugated IgG secondary antibodies for immunofluorescence experiments were purchased from Jackson ImmunoResearch (West Grove, PA). The secondary antibodies for Western blot analysis were horseradish peroxidase (HRP)-conjugated IgG and purchased from Santa Cruz Biotechnology, Inc. Anti-fade mounting medium (Vectashield) was from Vector Laboratories (Burlingame, CA). All the reagents for Western blot analysis were purchased from Bio-Rad Laboratories (Hercules, CA), and the chemiluminescence reagents for visualization from Pierce (Rockford, IL). Aprotinin and all other regents were from Sigma-Aldrich (St. Louis, MO). 

RNA Extraction and RT-PCR

The lacrimal gland was homogenized in extraction reagent (TRIzol; GibcoBRL, Grand Island, NY), according to the manufacturer’s protocol. As a positive control, total RNA was isolated from rat brain, which is known to synthesize the NT family of growth factors and receptors. Purified total RNA was used for complementary DNA (cDNA) synthesis by reverse transcription, as described in the manufacturer’s protocol (Promega). The cDNA was amplified by PCR, using specific primers ^{28 29 30} summarized in Table 1 , in a thermal cycler (PCR Sprint; Thermo Hybaid, Ashton, UK). Each PCR reaction consisted of 0.5 μM of each sense and antisense primer, 200 μM each of dNTP, 1.5 mM MgCl₂, 1.25 U of Taq polymerase, and 1 μL of cDNA. The conditions were: 5-minute hot start at 94°C, followed by indicated cycles (Table 1) of denaturation for 1 minute at 94°C, annealing for 1 minute at the indicated temperature (Table 1) , and extension for 1 minute at 72°C. The quality of cDNA was monitored using PCR with G3PDH primers (Clonetech Laboratories, Palo Alto, CA). The amplified products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. All RNA extractions and subsequent RT-PCR reactions were performed on three different animals. 

Electrophoresis and Immunoblot Analysis

Rat lacrimal glands were homogenized in RIPA buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA), containing proteinase inhibitors (100 μL/mL phenylmethylsulfonyl fluoride, 30 μL/mL aprotinin, and 100 nM sodium orthovanadate). After homogenization, the samples were centrifuged at 2000g for 30 minutes at 4°C to remove unbroken cells and nuclei. To detect NTs, proteins in the supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 15% acrylamide gels for NT, according to the method of Laemmli. ³¹ Proteins were then transferred by electrophoresis to nitrocellulose membranes, blocked in 5% dried milk in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween-20), and incubated with the indicated antibody for 1 hour at room temperature (1:200). Membranes were washed three times in TBST and incubated with HRP-conjugated anti-rabbit IgG (1:1000) for 1 hour at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Rat brain homogenate was used as a positive control. Negative controls included the omission of the primary antibody and preabsorption of the primary antibody with the corresponding peptide (10-fold excess and overnight incubation at 4°C) used for immunization. 

To detect the NTrs, a membrane fraction was isolated from rat lacrimal gland and brain homogenates prepared in homogenization buffer (30 mM Tris-HCl [pH 7.5], 10 mM EGTA, 5 mM EDTA, 1 mM dithiothreitol, and 250 mM sucrose) containing proteinase inhibitors (100 μL/mL phenylmethylsulfonyl fluoride, 30 μL/mL aprotinin, and 100 mM sodium orthovanadate). After homogenization the samples were centrifuged at 2000g for 15 minutes at 4°C. The pellet was then resuspended in the homogenization buffer and centrifuged at 100,000g for 1 hour at 4°C. The pellet containing the membrane fraction was resuspended in the homogenization buffer and proteins separated by SDS-PAGE on a 10% acrylamide gels, followed by Western blot analysis, as described earlier. 

Immunohistochemistry

For immunofluorescence microscopy the lacrimal gland was fixed in 4% formaldehyde in phosphate-buffered saline (PBS, 145 mM NaCl, 7.3 mM Na₂HPO₄, and 2.7 mM NaH₂PO₄ [pH 7.2]), for 4 hours at 4°C, rinsed in 5% sucrose dissolved in PBS, placed overnight in 30% sucrose dissolved in PBS at 4°C, embedded in OCT, and frozen. Cryostat sections (6 μm) were placed on slides (Colorfrost/Plus; Fisher Scientific, Pittsburgh, PA) and kept at −20°C until use. Lacrimal gland sections were then defrosted at room temperature for 1 hour, washed in PBS, and blocked in PBS containing 1% bovine serum albumin, 4% goat serum, and 0.2% to 0.3% Triton X-100. The NT antibodies (NGF, BDNF, NT3, and NT4) were used at a dilution of 1:500. The NTr antibodies (TrkA, TrkB, TrkC, and p75) were used at a dilution of 1:100. All antibodies were diluted in PBS containing 0.3%Triton X-100, and incubated for 48 hours at 4°C. The FITC-conjugated secondary antibody was then added (1:1000) for 1 hour at room temperature. After incubation, sections were washed in PBS and covered with coverslips. Sections were viewed and photographed under a microscope (Eclipse E800; Nikon, Melville, NY) equipped with a digital camera (Spot; Diagnostic Instruments, Inc., Sterling Heights, MI). Negative control experiments included the omission of the primary antibody and preabsorption of the primary antibody with the corresponding peptide (10-fold excess and overnight incubation at 4°C) used for immunization as previously described. ^{32 33 34}  

Results

Detection of NTs and NTr by RT-PCR

In a first series of experiments, we used RT-PCR to identify the types of NTs and NTrs present in the lacrimal gland. As shown in Figure 1 , cDNA of the expected sizes for NGF, BDNF, NT3, and NT4 were detected in the lacrimal gland. Figure 2 shows that cDNA at the expected sizes for TrkA, TrkB, TrkC, and p75 were also detected in the lacrimal gland. With rat brain cDNA, the positive control, both the NTs and NTrs were detected at the same size as in the lacrimal gland. 

No bands were obtained when the PCR was performed in the absence of cDNA. G3PDH primers were also used to ensure the integrity of the cDNA (data not shown). In both Figures 1 and 2 , three individual lanes of lacrimal gland cDNA are shown, with each lane representing a single animal. 

Detection of NT and NTr by Western Blot Analysis

The data shown in Figures 1 and 2 suggest that NGF, BDNF NT3, NT4 NTs, and TrkA, TrkB, TrkC, p75 NTrs are present in lacrimal gland. To determine whether these mRNAs are translated into their corresponding proteins, we performed Western blot analysis (Figs. 3 and 4) . Figure 3 shows the presence of NGF, BDNF, NT3, and NT4 in the homogenate from the lacrimal gland. Brain homogenate was used as the positive control. The NTs were detected in the lacrimal gland, and the positive control as a single band at molecular masses between the apparent molecular masses of 26 and 50 kDa, suggesting that NTs are present as dimers similar to other published studies. ³⁵  

Negative control experiments included the omission of the primary antibody and preabsorption of the primary antibody with the corresponding peptide (10-fold excess and overnight incubation at 4°C) used for immunization. No bands were detected under these conditions (data not shown). 

Figure 4 shows the presence of NTrs in membrane fractions prepared from homogenates of the lacrimal gland and brain, the positive control. TrkA was detected in the lacrimal gland and brain at the apparent molecular mass of 145 kDa, corresponding to the full-length Trk receptor protein. Rat brain also showed other bands at apparent molecular masses of 115 and 97 kDa illustrating the variable degree of glycosylation that can occur during the transport of the receptor to the cell membrane. ¹⁷ This has been described for different tissues. ^{35 36} The NTr TrkB was detected as a single band, at the apparent molecular mass of 140 kDa in the lacrimal gland, whereas the brain showed, in addition to this band, another band at the apparent molecular mass of 130 kDa. The band at 140 kDa matches the molecular mass of the full-length TrkB receptor, whereas the other band in the brain represents a different degree of glycosylation acquired by this receptor. A single band was detected at the apparent molecular mass of 140 kDa for TrkC in the lacrimal gland. This receptor was also detected in the brain, similar to the other NTr, TrkB, with an additional band located at 130 to 135 kDa, indicating variable glycosylation of this receptor. The p75 receptor was also present as a single band at the expected 75 kDa in both lacrimal gland and brain. In the panels of Figures 3 and 4 , three individual lanes of lacrimal gland homogenates are shown, with each lane representing a single animal. 

Cellular Location of NT and NTr

Using immunofluorescence microscopy, we determined the cellular location of the NTs and NTrs in the lacrimal gland. NGF, BDNF, NT3, and NT4 were each present on myoepithelial cells (Fig. 5) . NGF was also observed in a punctate cytoplasmic distribution on intracellular membranes, possibly the endoplasmic reticulum or Golgi apparatus in the acinar cells (Fig. 5A) . BDNF and NT4 were also present in the acinar cell cytoplasm in a diffusely distributed pattern (Figs. 5B 5D) . In addiction NGF, BDNF and NT3 were present in ductal cells (Figs. 5A 5B 5C) . Positive immunoreactivity was also occasionally detected in the endothelial cells of the blood vessels (data not shown). No staining was observed when the primary antibodies were omitted (data not shown). 

TrkA, TrkB, and TrkC appeared to be localized primarily on the myoepithelial cells (Fig. 6) . TrkB and TrkC in addition showed staining on ductal cell membranes (Figs. 6B 6C) . Immunoreactivity for p75 appeared to be only in the myoepithelial cell membranes (Fig. 6D) . The positive immunofluorescence was not detected when the primary antibodies were omitted. Another control experiment, the use of preadsorbed antibodies with a 10-fold excess of the immunizing antigen peptide, was also negative (data not shown). 

Discussion

Using three different techniques: RT-PCR, Western blot analysis, and immunofluorescence microscopy, we showed the presence of mRNA transcripts, the translated corresponding proteins, and localization of multiple NTs and NTrs in the adult rat lacrimal gland. All three methods showed the presence of NGF, BDNF, NT3, and NT4 and TrkA, TrkB, TrkC, and p75. Whereas Nguyen et al. ³⁷ have previously shown the presence of NGF and p75 mRNA in human lacrimal gland, this study extends those findings to show mRNA presence with the translated products, and the location of multiple NTs and NTrs in rat adult lacrimal gland tissue. The lacrimal gland is then similar to the cornea, retina, brain, and many other tissues in which multiple NTs and NTrs have been shown to be present. Furthermore, our results support studies indicating that non-neuronal cells are able to synthesize and express NTs and NTrs. ^{38 39 40}  

It is possible that, similar to other tissues where NTs have a well-studied roles, NTs in the lacrimal gland can help maintain the survival and differentiated phenotype of the sensory and autonomic neurons that have been shown to innervate this tissue ^{41 42} and/or could promote the neural innervation. In this scenario, NTs such as NGF and BDNF may be secreted across the basolateral membranes of lacrimal gland acinar cells. BDNF, NT3, and NT4 could also be released by the myoepithelial cells, in that we have shown them to be present in these cells. Secreted NTs could then have an effect on the nerves. In addition, these growth factors could affect acinar cells and also the myoepithelial cells in a paracrine and/or autocrine fashion. The NTs secreted from acinar and myoepithelial cells may have multiple effects in the target tissues (nerves, acinar cells, and myoepithelial cells). This supports the increasing number of studies that identify NTs as critical growth factors in development, survival, aging, and also injury response of many cell types and tissues. Alternatively, as has been well described, the NTs can also be secreted across the apical membrane of acinar cells into the ductal lumen and hence into the tears. NTs, in fact, are detectable in the tears. ^{41 42} This suggests that it is possible for NGF, BDNF, NT4, and probably NT3 to reach the anterior ocular surface and have a role in a wide spectrum of biological events on the conjunctival and corneal tissue. Ductal cells may also have a role in modifying the final concentrations of these NTs on the ocular surface by secreting or absorbing electrolytes and water. This regulating action could be dependent on TrkB and TrkC receptors, which are also expressed on ductal cells. 

It is interesting also to observe, that p75, the NT low-affinity receptor that is able to increase the binding specificity of NTs to NTrs but is also able to bind all the other NTs individually, appeared to be present only on myoepithelial cell membranes (Fig. 6D) . These cells then might be able to use NTs released from other cell types through the expression of p75 with the other NTrs. Because they express NTs and NTrs, myoepithelial cells could secrete NTs that then activate the NTrs, similar to the effect of NTs in other tissues. ^{45 46}  

The role of myoepithelial cells in the lacrimal gland is still unknown, and it is hypothesized that they are involved in contraction of the acinar cells. Thus, it is possible that this function of the myoepithelial cells may be regulated by the NTs through their NTrs, as NGF does in other tissues where it is known to stimulate contraction in a variety of cell types in vivo. ^{36 47}  

As many others researchers believe, the components of the ocular surface (the cornea, the conjunctiva, the accessory and main lacrimal glands) and the interconnecting innervation between these structures act as a morphofunctional unit and alterations in this feedback could lead to dry eye. ^{48 49} The nerves in the cornea and conjunctiva are part of an afferent pathway to the central nervous system from where efferent nerves reach the lacrimal gland. The presence of NTs and NTrs in the lacrimal gland may represent a key point in the network created by the ocular surface, the nerves, the brain stem and the lacrimal gland. Reflex activation of efferent nerves in the lacrimal gland may cause a release of NTs from this gland. NTs released from myoepithelial cells and from basolateral side of acini may then activate NTr located on myoepithelial and ductal cells. The NTs released from the apical side of acini may appear in tears and activate NTrs found in the conjunctiva (Ghinelli E, Rios JD, Zoukhri D, Johansson J, Dartt DA, ARVO Abstract 3172, 2002) and cornea. ²⁵  

In conclusion, in the present study, NGF, BDNF, NT3, and NT4 NTs and TrkA, TrkB, TrkC, and p75 NTrs were expressed by the lacrimal gland tissue, suggesting that these proteins may play a role in the health and maintenance of the lacrimal gland, may regulate secretion from this tissue, and may be secreted from the lacrimal gland and thus affect the functioning of the cornea and conjunctiva. 

 Supported by National Eye Institute Grant EY06177.

 Submitted for publication January 14, 2003; accepted March 4, 2003.

 Disclosure: E. Ghinelli, None; J. Johansson, None; J.D. Ríos, None; L.L. Chen, None; D. Zoukhri, None; R.R. Hodges, None; D.A. Dartt, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Sequences and PCR Parameters.

NT/NTr	Oligonucleotide Sequence			Position	Size (bp)	AT	Acc. No.	Cycles (n)
NGF	Sense	5′-cggcgtacaggcagaaccgtacacag-3′	335–360	410	54	M35075	30
	Antisense	5′-gtgtgggttggagataagaccacagccacag-3′	714–744
BDNF	Sense	5′-cagtggacatgtccggtgggacggtc-3′	545–570	533	60	NM_012513	35
	Antisense	5′-gttgtggtttgttgccgttgccaagaa-3′	1051–1077
NT3	Sense	5′-gcaacagacacagaactacta-3′	331–351	232	62	M34643	30
	Antisense	5′-gcctgtgggtgaccgacaagt-3′	542–562
NT4	Sense	5′-gtacttcttcgagacgcgctgc-3′	477–498	135	62	M86742	37
	Antisense	5′-gcccgcacataggactgtttagc-3′	589–611
TrkA	Sense	5′-cgtggaacagcatca-3′	946–960	339	52	M85214	30
	Antisense	5′-gacactaacagcacatcaag-3′	1265–1284
TrkB	Sense	5′-ggacacgcactctgactgactggcact-3′	71–97	713	65	M55293	35
	Antisense	5′-tcctgcagcgtcgggggtgacccgctc-3′	757–783
TrkC	Sense	5′-atgtgggctccgtgctggcttgccctgcaa-3′	125–154	366	62	L03813	30
	Antisense	5′-accggctcaccacactctcctggcagctct-3′	461–490
p75	Sense	5′-gagggcacatactcagacgaagcc-3′	567–590	663	58	X05137	30
	Antisense	5′-ggttaccagcctgaacatatagac-3′	1206–1229

Figure 1.

 

RT-PCR analysis of RNA extracted from lacrimal gland homogenate (L.G.). Each LG lane represents a different animal, with brain homogenate (B) used as the positive control. Brain and lacrimal gland expressed NGF, BDNF, NT3, and NT4 NTs. L, DNA ladder.

Figure 2.

 

RT-PCR analysis of RNA extracted from lacrimal gland homogenate (L.G.). Each LG lane represents a different animal, with brain homogenate used as the positive control (B). Brain and lacrimal gland expressed TrkA, TrkB, TrkC, and p75 NTrs. L, DNA ladder.

Figure 3.

 

Western blot analysis of NGF, BDNF, NT3, and NT4 NT proteins in homogenized lacrimal gland tissue (L.G.), with brain (B) loaded as the positive control. Each LG lane represents a different animal.

Figure 4.

 

Western blot analysis of TrkA, TrkB, TrkC, and p75 NTrs proteins in membrane fractions prepared from homogenates of the lacrimal gland tissue (L.G.) and brain (B), loaded as the positive control. Each LG lane represents a different animal.

Figure 5.

 

Immunolocalization of NGF (A), BDNF (B), NT3 (C), and NT4 (D) NT proteins in the lacrimal gland acini, ductal cells (arrowheads), and on myoepithelial cell membranes (arrows). Similar results were observed in at least three other animals. Magnification, ×200.

Figure 6.

 

Immunolocalization of TrkA (A), TrkB (B), TrkC (C), and p75 (D) NTr proteins in the lacrimal gland acini, ductal cells (arrowheads), and on myoepithelial cell membranes (arrows). Similar results were observed in at least three other animals. Magnification, ×200.

The authors thank Alessandro Lambiase and Stefano Bonini (University of Rome Campus bio Medico, Rome, Italy) and David A. Sullivan (Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts) for helpful discussions and advice.