April 2004
Volume 45, Issue 4
Free
Immunology and Microbiology  |   April 2004
Murine Ocular Heparanase Expression before and during Infection with Pseudomonas aeruginosa
Author Affiliations
  • Richard S. Berk
    From the Departments of Immunology and Microbiology,
  • Zhong Dong
    Urology,
  • Sarah Alousi
    Pathology, and
  • Mary Ann Kosir
    Surgery, Wayne State University School of Medicine, Detroit, Michigan; the
    Department of Surgery, Veterans Administration Medical Center, Detroit, Michigan; and the
  • Yuying Wang
    Surgery, Wayne State University School of Medicine, Detroit, Michigan; the
    Department of Surgery, Veterans Administration Medical Center, Detroit, Michigan; and the
  • Israel Vlodavsky
    Department of Oncology, Hadassah University Hospital, Jerusalem, Israel.
Investigative Ophthalmology & Visual Science April 2004, Vol.45, 1182-1187. doi:10.1167/iovs.03-0589
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      Richard S. Berk, Zhong Dong, Sarah Alousi, Mary Ann Kosir, Yuying Wang, Israel Vlodavsky; Murine Ocular Heparanase Expression before and during Infection with Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2004;45(4):1182-1187. doi: 10.1167/iovs.03-0589.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To demonstrate the constitutive expression and regulation of heparanase (heparan sulfate endoglycosidase) in the normal mouse eye and in mice intracorneally infected with Pseudomonas aeruginosa.

methods. Naïve (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and corneal heparanase gene and protein expression were detected by semiquantitative RT-PCR and immunoblot analysis. Immunohistochemistry was also applied to characterize corneal heparanase in naïve mice.

results. Heparanase mRNA and protein expression were detected in uninfected corneas of C57BL/6J mice. Immunohistochemical studies indicated heparanase protein expression was primarily in the corneal epithelium before corneal infection and was also in the corneal stroma after infection. Immunohistochemical studies of uninfected and infected whole eyes of naïve mice indicated heparanase protein expression in most layers of the retina, but the expression did not appear to be upregulated during corneal infection. Staining was most intense in the inner photoreceptor layer of the retina.

conclusions. Heparanase was constitutively expressed in both the corneal epithelium and several retinal layers before intracorneal infection with P. aeruginosa. Temporal upregulation of corneal heparanase protein expression was detected in naïve mice during infection, most likely due to heparanase positive infiltrating cells, but the protein was not upregulated in corneas from immunized mice because they had a lower inflammatory response, associated with the restoration of corneal clarity. There did not appear to be temporal upregulation of heparanase expression in the retina of infected mice, as determined by immunohistochemistry.

Heparan sulfate proteoglycans, which are components of the cell surfaces, extracellular matrix (ECM), and basement membranes, interact with many proteins through their heparan sulfate side chains. Endoglycosidic cleavage by heparanase (endo-β-d-glucuronidase) is thought to affect a variety of biological processes. Heparanase expression in mammalian tissues has been well established for several years, but the molecular properties and substrate specificity of the enzyme(s) have been difficult to study and have remained elusive. In addition, several attempts to purify heparanase(s) to homogeneity have been difficult due to low levels, instability, and lack of a convenient quantitative assay. Heparanases have been found in a number of different sources and species and appear to have a wide range of molecular masses, adding to the confusion. For example, human platelet heparanase has been reported to have a molecular mass ranging from 8 to 137 kDa, 1 2 3 whereas a 98-kDa heparanase was purified from mouse melanoma cells and partially sequenced. 4 A family of four heparanases with different physical characteristics have been purified from Chinese hamster ovary (CHO) cells and rat liver. 5 Their molecular masses range from 37 to 48 kDa. The most abundant of these enzymes has a molecular mass of 40 kDa and is highly homologous with the N terminus of the 80-kDa proteins ezrin, radixin, and moesin. 6 Immunoblot analyses with a monoclonal antibody raised against human neutrophil heparanase recognized a 96-kDa band which has a molecular mass similar to that of human and mouse melanoma heparanase. 7  
One of the best studied heparanases is Hpa1, which is a latent, 65-kDa glycoprotein expressed primarily in the placenta and cells of the immune system. 8 9 10 Its activated form may exist as a heterodimer composed of an 8-kDa N-terminal subunit and a 50-kDa C-terminal subunit, resulting from proteolytic processing and linker region excision of the latent full-length protein. 11 Vlodavsky et al., 12 Hulett et al., 13 and Kussie et al. 14 have reported the cDNA and derived amino acid sequence for the 50-kDa enzyme. An Hpa2 has recently been described that codes for three proteins generated by alternative splicing of the Hpa2 transcript. 15 Hpa2 differs from Hpa1, in that it lacks a signal peptide sequence and has different tissue distributions and possibly different cellular locations. 15  
The physiologic functions of heparanases may be manifold. For example, they can contribute to the remodeling of the ECM and basement membrane, which is a prerequisite to the angiogenesis cascade and to the egress of metastatic tumor cells and other types of blood-borne cells from the vasculature, 12 making it a promising target for drug development. They may also mediate the release of subendothelial-bound lipoprotein lipase and may modulate the coagulation process. 16 Up to now, there appear to be no publications on heparanase expression in the eye or during ocular infection. The relationship between heparanase expression and heparan sulfate proteoglycans may play an important role in host–parasite relationships, inflammation, angiogenesis, and bacterial virulence, due to the shedding of syndecans. Heparanase along with other enzymes, may play a major role in regulating the turnover of the ECM and basement membrane in both normal and infected eyes. Consequently, the purpose of the present study, is to establish the heparanase (hpa1) gene and protein expression in the normal murine cornea, before and during corneal infection with Pseudomonas aeruginosa, by using semiquantitative RT-PCR, Western blot analysis, and immunohistochemistry. In addition, immunohistochemistry was used to demonstrate retinal heparanase expression which was not affected by corneal infection. 
Methods
Bacteria
Stock cultures of P. aeruginosa 19660 (ATCC, Manassas, VA) were stored at 4°C on tryptose agar slants (Difco Laboratories, Detroit, MI) and were used for the inoculation of 50 to 75 mL of broth medium containing 5% peptone (Difco Laboratories) and 0.25% trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD). Strain 19660 is hemolytic and lecithinolytic and produces exotoxin A, alkaline protease, and elastase under appropriate culture conditions. Cultures were grown on a rotary shaker at 37°C overnight, centrifuged at 6000 rpm at 4°C for 10 minutes, washed with normal saline (Travenol Laboratories, Cambridge, MA), and diluted to a concentration of 2 × 1010 colony-forming units per milliliter. A standard curve was developed to relate viable counts to optical density at 440 nm. 
Infection of Animals
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Age-matched naïve and immunized C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME), each weighing 22 to 26 g, were infected at 14 weeks of age. Before infection, they were lightly anesthetized with ether and placed beneath a stereoscopic microscope. The corneal surface was then gently incised with three 1-mm incisions with a sterile 26-gauge needle, with care taken not to penetrate the anterior chamber or to damage the sclera. A bacterial suspension (5 μL) containing 108 colony–forming units was topically delivered onto the wounded cornea using a micropipette with a sterile disposable tip. Controls consisted of scratched and unscratched mice that were uninfected. Mice were examined 24 hours later to verify infection. Naïve C57BL/6J mice have previously been classified as susceptible because corneal clarity is not restored in them, whereas immunized mice are considered resistant because corneal clarity is restored within a few days. 17 18 Immunization of the mice was begun at 6 weeks of age by administration of 0.1 mL 106 to 107 heat-killed P. aeruginosa 19660 intraperitoneally weekly for 4 weeks, and then the mice were rested for 4 weeks before corneal infection. 
Corneal Sample Collection and Processing
At selected time points after infection, mice were killed and corneas were excised. Individual samples for reverse transcriptase–polymerase chain reaction (RT-PCR) and immunoblot analysis consisted of 12 pooled corneas per time period. Immediately after isolation, corneas were rinsed in sterile saline and then were processed for the purposes of the different assays. Control mice were treated similarly. 
Semiquantitative RT-PCR
Total RNA was isolation from the harvested corneas with extraction reagent (TRIzol; Invitrogen-Gibco, Grand Island, NY), according to the manufacturer’s instructions. The total RNA was dissolved in water treated with diethyl pyrocarbonate (DEPC), and the concentration was measured with a spectrophotometer (model UV-1601; Shimadzu, Kyoto, Japan). All the reagents needed for RT-PCR were purchased from Perkin Elmer (Boston, MA) except Taq DNA polymerase and dNTP mixture which were purchased from Invitrogen-Gibco. 
RT-PCR was performed as previously described. 19 Briefly, reverse transcription was performed in 0.65-mL RNase-free tubes under optimized conditions in a DNA thermal cycler (model 480; Perkin Elmer). An equal amounts of total RNA (500 ng) from each sample was used for this reaction. The whole product of reverse transcription in each tube was amplified by polymerase chain reaction. Cycle parameters were a 1-minute melting step at 95°C, a 1-minute annealing step at 55°C, and a 2-minute extension at 72°C. Thirty-five cycles were selected for amplification of the heparanase gene, and 25 cycles were chosen for the GAPDH gene, based on the experiments that tested the linear range of amplification with different cycles. The specific primers for heparanase and GAPDH were designed according to their mRNA sequences available in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). The primers of mouse heparanase (5′-TTT GCA GCT GGC TTT ATG TG-3′ and 5′-GTC TGG GCC TTT CAC TCT TG-3′) amplified a 207-bp product. There were two negative controls: one without reverse transcriptase and the other one without specific primers. GADPH was also amplified and used as an internal control for the comparison of all time point target genes. Finally, the amplified genes were resolved by 1% agarose gels and revealed by ethidium bromide staining. 
Immunoblot Analysis
Corneal samples were homogenized as described by Brown et al. 20 After homogenization in 200 μL Tris-HCl (pH 7.4; 50 mM), containing 10 mM CaCl2, 1% Triton X-100 and a cocktail of protease inhibitors, the samples were centrifuged at 9000 rpm at 4°C for 30 minutes. The concentrations of the total protein were measured with the bicinchoninic acid (BCA) protein assay. 21 Equal amounts of individual samples (12 μg) were mixed with 5 μL of 4× sample loading buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 40% glycerol, and 0.02% bromphenol blue), containing β-mercaptoethanol, and boiled for 5 minutes. The samples and a prestained molecular mass marker (Bio-Rad, Cambridge, MA) were electrophoresed on 12% SDS gels and subsequently transferred to nitrocellulose membranes. The membranes were blocked for 30 minutes (Tris-buffered saline, containing 0.5% Tween 20, 3% nonfat milk, and 2% bovine serum albumin; Blotto; Santa Cruz Biotechnology, Santa, Cruz, CA) and then incubated for 2 hours at room temperature with the specific primary antibody, a polyclonal antibody directed to four fractions of the recombinant 50-kDa human heparanase that reacts with human and mouse Hpa1 heparanase. In Western blots, the antibody recognizes primarily the active form of the enzyme (50 kDa) and to a much lower extent the 65-kDa latent form (ImClone Systems, New York, NY). Samples without primary antibody treatment were processed as negative controls or treated with an irrelevant antibody of the same isotype or normal serum. Afterward, the blots were incubated with secondary antibody conjugated with horseradish peroxidase (0.5 μg/mL; Roche Diagnostics, Indianapolis, IN) at room temperature for 1 hour. Finally, the blots were developed by the chemiluminescence kit (Amersham, Arlington Heights, IL), and heparanase was visualized as dark bands. 
Immunohistochemistry
Immunohistochemistry was performed using the procedure of Vlodavsky et al. 12 with minor modifications. Briefly, 5-μm sections were deparaffinized and rehydrated. The tissues were then denatured for 3 minutes in a microwave oven in 0.01 M citrate buffer (pH 6.0). Blocking steps included incubation in 3% hydrogen peroxide in methanol and 5% goat serum, followed by two washes in phosphate-buffered saline. Sections were incubated with the rabbit polyclonal antibody to mouse and human recombinant Hpa1 diluted 1:100 in PBS, or with Dulbecco’s modified Eagles’ medium supplemented with 10% horse serum as a control, followed by incubation with horseradish peroxidase–conjugated goat anti-rabbit IgG (Vector Labs, Burlingame, CA). Color was developed using a 3,3′-diaminobenzidine (DAB) kit (SK4100; Sigma-Aldrich) for 10 minutes followed by counterstaining with Mayer’s hematoxylin. Heparanase-negative tissues stained blue, and heparanase-positive tissues and infiltrating cells stained orange to reddish brown. 
Results
Semiquantitative RT-PCR was initially performed to determine whether corneal homogenates prepared from naïve and immunized mice contained mRNA for heparanase and whether heparanase expression was modulated at the transcription level by corneal infection. The corneas were harvested on day 0 (uninfected control) and on days 1, 3, 5, and 8 after infection, which represented the overall corneal inflammatory response in naïve and immunized mice. 22 23 24 25 By day 8, the unimmunized mice exhibited extensive keratitis, whereas the immunized mice showed restored corneal clarity. As depicted in Figure 1 , there was constitutive expression of heparanase mRNA in the corneas before infection of both naïve and immunized mice. When mice were infected intracorneally with P. aeruginosa, mRNA expression in the naïve mice at days 1 and 3 remained relatively unchanged, but expression increased above the day 0 control level by days 5 and 8 after infection. In immunized mice corneal clarity was restored, 17 18 mRNA expression at time 0 remained relatively constant and intense at all time points (data not shown). Amplified heparanase samples were sequenced, and the results confirmed the specificity for the primers used for RT-PCR (data not shown). No differences in heparanase mRNA expression were found between uninfected scratched and unscratched corneas in both naïve and immunized mice, indicating that corneal abrasion did not affect the mRNA expression (data not shown). No transcripts were observed in negative control samples (samples not treated with reverse transcriptase or amplified without specific primers; data not shown). 
To confirm heparanase expression at the protein level on a temporal basis, immunoblots were performed in duplicate (Fig. 2) on corneal extracts from both naïve and immunized mice over an 8-day postinfection period. Extracts from uninfected corneas from naïve mice exhibited a faint band at approximately 65 kDa which most likely represented the latent enzyme before activation, but a band was not detected at 50 kDa (activated enzyme) until after infection. Extracts from naïve mice infected with P. aeruginosa exhibited bands at approximately 50 kDa at days 3, 5, and 8 (Fig. 2) . Faint bands were also detected at 65 kDa. As expected, the 50-kDa bands were not detected in extracts from immunized mice, indicating a dampened inflammatory response coincident with the rapid restoration of corneal clarity. Faint bands observed below 50 kDa may have been due to overprocessing of the latent enzyme, other heparanases that have partial homology with Hpa1, or nonspecific binding. 5 15  
Immunohistochemical staining for corneal Hpa1 protein from normal and infected corneas of naïve mice was conducted, and the results are shown in Figure 3 . Controls consisting of sections at days 0 and 6 after infection were stained with an irrelevant IgG or normal serum or were unstained (Figs. 3A 3C) . No positive Hpa1 immunostaining was detected in the control samples. Cellular infiltrates were not detected in the stroma of the uninfected corneas, but were present in the infected mice. When corneas from uninfected mice were stained with the Hpa1 antibody, positive staining was primarily in the corneal epithelium (Fig. 3B) . Positive immunostaining of both the corneal epithelium and stroma was seen 1 day after infection (not shown) and 6 days after infection—the time at which the response normally peaks and after which it normally diminishes. 22 23 24 The cellular infiltrate consisting of primarily polymorphonuclear (PMN) leukocytes in the stroma stained positive for Hpa1 (Figs. 3D 3E) . Figure 3D shows that the vascular endothelium of blood vessels in the cornea stained strongly for heparanase. Similar results were observed in the corneas of immunized mice, but there were fewer Hpa 1–positive infiltrating cells (data not shown). 
Because corneal heparanase expression was constitutively present in normal, uninfected mice, we set out to determine whether the retina, iris, and lens also express heparanase constitutively and whether expression was upregulated during the infection. Immunohistochemical staining on sectioned whole eyes was performed both before and after infection for 6 days. Positive immunostaining appeared in retinas from both uninfected and infected eyes, indicating constitutive protein expression. Surprisingly, there did not appear to be any major discernible difference in heparanase expression between retinal sections from uninfected and infected mice over a 6-day period of infection. Therefore, the immunohistochemical results in Figures 4A and 4B represent uninfected retinas and indicate intense retinal inner photoreceptor staining with very faint staining of the outer photoreceptor layer. Positive staining of various intensities was seen throughout the retinal layers (i.e., inner nuclear, inner and outer plexiform layers, ganglion cells, amacrine cells, inner limiting membrane, optic nerve fiber layer, ganglion layer, blood vessels in the outer plexiform layer and possibly the horizontal cells). The outer nuclear area appeared to stain faintly, but there was perikaryal or cytoplasmic staining of this layer which was Hpa-1 positive (Figs. 4B 4C) . Negative control samples did not exhibit positive immunostaining in the retina. The lens showed little or no staining for Hpa1, whereas faint staining was detected in the iris (not shown). Because of the nature of the pigmented epithelium of the choroid, it was difficult to determine whether positive staining occurred. Figure 4D depicts the positive staining of the ganglion cells. 
Discussion
The inflammation caused by P. aeruginosa corneal infection starts with a series of local host reactions such as edema, leukocyte infiltration, and angiogenesis. Corneal ulceration may occur and corneal perforation may eventually take place as the result of stromal dissolution. 22 23 24 Both host and bacterial proteases may contribute to the degradation of the ECM and basement membrane. We recently reported the expression of corneal matrix metalloproteinases (MMPs) and the constitutively expressed membrane type (MT)-MMPs, plasminogen activators, and cathepsins. 19 25 26 27 28 These were upregulated during the inflammatory response seen in naïve mice, whereas in immunized mice corneal clarity was restored and lower and briefer enzyme expression was exhibited. The degree of enzyme expression correlated with the inflammatory response. Thus, both naïve and immunized mice have provided useful in vivo models to study the relationship between the expression of various corneal enzymes and corneal destruction, as well as wound healing during corneal infection. 
In the present study, we investigated the ocular expression of a novel enzyme termed heparanase that has never been described in the eye before. It is an endo-β-d-glucuronidase that degrades heparan sulfate. We used semiquantitative RT-PCR, immunoblot analysis, and immunohistochemistry to demonstrate the constitutive expression of heparanase in uninfected and infected corneas. In addition, immunohistochemistry was used to demonstrate the constitutive expression of heparanase in several retinal layers. Furthermore, intracorneal infection of naïve mice with P. aeruginosa indicated temporal upregulation of corneal heparanase expression, as determined by immunoblot analysis, whereas corneal extracts from immunized mice showed stable heparanase expression, signaling a reduced inflammatory response, as previously noted with various proteases. 19 25 26 27 28 However, there did not appear to be a substantive increase in heparanase expression in the retina during corneal infection as determined by immunohistochemistry. Heparanase expression was limited to the corneal epithelium of uninfected mice, but expression in infected mice was noted in the stroma as well, resulting from cellular infiltration of heparanase-positive cells. Previous corneal studies have indicated that the initial cellular infiltrates during infection consist primarily of PMN leukocytes followed by macrophages. 22 High levels of heparanase activity have previously been found in both cell types and platelets, and T and B cells. 29  
The relationship between heparanase expression and heparan sulfate proteoglycans may play an important role in host parasite relationships and inflammation in particular. Binding of heparan sulfate to interleukin-8 has been shown to enhance neutrophil chemotaxis, but elastase and cathepsin G from stimulated neutrophils were inhibited by heparan sulfate and heparin. 30 31 In addition, P. aeruginosa, in particular, produces the potential virulence factor LasA, which enhances the shedding of the transmembrane heparan sulfate proteoglycan syndecan-1, in vitro and in vivo. 32 This results in enhancement of bacterial virulence in newborn mice, whereas mice deficient in syndecan-1 resist P. aeruginosa pulmonary infection. 33 Inhibition of syndecan-1 shedding also prevents lung infection. Consequently, cell surface heparan sulfate proteoglycans are important host determinants for microbial pathogenesis. 
Heparanase expression can result in the release of heparan-sulfate–bound angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) from the ECM and basement membrane. 9 12 Heparan sulfate proteoglycans are required for the interaction of bFGF and VEGF ligands and their receptors. However, their role in regulating glutamate leucine arginine (ELR)-chemokine signaling and biological functions is not well understood and requires further study. The generation of heparan sulfate fragments results in the potentiation of bFGF receptor binding, dimerization, and signaling. 34 A role in vascularization is also suggested by heparanase staining of the blood vessels in the retina, as also demonstrated in other tissues. 35 In addition, heparanase expression can release subendothelial bound lipoprotein lipase. 16 36  
One of the physiological functions of heparanase in the eye may be the regulation of heparan sulfate turnover. Heparan sulfate is an important component of the ECM and the vascular basal lamina, which functions as a barrier to the extravasation of inflammatory and metastatic cells. Cleavage of heparan sulfate through the heparanase activity of resident and invading cells may play a role in the disassembly of the ECM and basal lamina and thus facilitate cell migration. In addition, plasminogen activator has been shown to be activated by treatment with the chemokine CTAP-III, which is thought by some investigators to express heparanase activity. 2 37 This suggests that the ECM–resident plasminogen activating enzyme is sequestered in an active form by the subendothelial ECM. 38 Previous studies from our laboratory have demonstrated the corneal expression and upregulation of plasminogen activators during corneal infection with P. aeruginosa. 27 Thus, heparanase expression may directly and indirectly play a role in the overall inflammatory process during corneal infection. 
The main source of heparan sulfate at the cell surface is the syndecan family of four transmembrane proteoglycans. Similar to cell surface proteins, they can be converted into soluble molecules by enzymatic shedding of their ectodomains. 39 They are highly expressed in neural tissues and have been highly expressed in nerve fiber-rich layers of the retina at early postnatal stages. N-syndecan immunoreactivity was observed by Inatani et al. 40 in the ganglion cell layer, by in situ hybridization. Consequently, retinal heparanase may play a pivotal role in the development and turnover of retinal heparan sulfate proteoglycans. In addition, the four members of the syndecan family of transmembrane heparan sulfate proteoglycans are found within the trabecular meshwork/Schlemm’s canal system and the ciliary body of normal human eyes. 41 Their presence within the ciliary muscle may indicate participation in regulating aqueous outflow from the anterior chamber (Filla MS, et al. IOVS 2002;43:ARVO E-Abstract 1025). 
Heparanase Hpa1 mRNA is normally found in the placenta 8 and activated cells of the immune system, 10 41 but up to now it has not been associated with ocular tissue. The latent form of the enzyme has a molecular mass of approximately 65 kDa before proteolytic activation. The active enzyme has been postulated to be in the area of 50 kDa. 11 In the present study, we demonstrated the expression of both the latent 65- and 50-kDa activated enzyme as determined by immunoblot analysis with an Hpa1 specific polyclonal antibody made to the recombinant enzyme. 
At the present time, the role(s) of heparanase in the eye is unknown and requires further study. Nevertheless, we believe that this is the first description of ocular heparanase expression, and we have partially characterized it prior to and during corneal infection with P. aeruginosa. Given the constitutive expression of the enzyme in both the cornea and retina, it is possible that the enzyme in conjunction with its heparan sulfate substrate may play a role in tissue destruction and regeneration, diapedesis, angiogenesis, and regulation of heparan sulfate turnover. These putative functions will be the subject of future studies. 
 
Figure 1.
 
Semiquantitative RT-PCR determination of heparanase mRNA expression in the mouse corneas before and during infection with P. aeruginosa. Naïve mice were infected with P. aeruginosa and total RNA samples were prepared from the pooled corneas harvested over an 8-day period. Equal amounts of total RNA (500 ng) from individual samples were used for RT-PCR. Specific primers for mouse heparanase were used to amplify the target genes that were stained by ethidium bromide as bright bands in the dark background. The GAPDH gene was also amplified and used as an internal control.
Figure 1.
 
Semiquantitative RT-PCR determination of heparanase mRNA expression in the mouse corneas before and during infection with P. aeruginosa. Naïve mice were infected with P. aeruginosa and total RNA samples were prepared from the pooled corneas harvested over an 8-day period. Equal amounts of total RNA (500 ng) from individual samples were used for RT-PCR. Specific primers for mouse heparanase were used to amplify the target genes that were stained by ethidium bromide as bright bands in the dark background. The GAPDH gene was also amplified and used as an internal control.
Figure 2.
 
Expression of heparanase protein in the mouse corneas before and during infection as determined by immunoblot analysis. Naïve and immunized mice were infected with P. aeruginosa, and the eyes were harvested over an 8-day infection period. Equal amounts of total protein (12 μg) of each time-point sample were loaded for electrophoresis. A rabbit polyclonal antibody raised to recombinant heparanase fractions was used for immunoblot analysis. MW, molecular weight; r-Hp, recombinant heparanase.
Figure 2.
 
Expression of heparanase protein in the mouse corneas before and during infection as determined by immunoblot analysis. Naïve and immunized mice were infected with P. aeruginosa, and the eyes were harvested over an 8-day infection period. Equal amounts of total protein (12 μg) of each time-point sample were loaded for electrophoresis. A rabbit polyclonal antibody raised to recombinant heparanase fractions was used for immunoblot analysis. MW, molecular weight; r-Hp, recombinant heparanase.
Figure 3.
 
Immunohistochemistry of the cornea. Uninfected and infected corneas from naïve mice were stained with normal serum, irrelevant IgG (control samples), or Hpa1 antibody, to detect the heparanase protein (days 0, 1, and 6) after infection. (A) Section of normal control cornea not stained with Hpa1 antibody. (B) Uninfected cornea stained with the Hpa1 antibody. Intense staining of the corneal epithelium with faint staining of the stroma is depicted. CE, corneal epithelium; CS, corneal stroma. (C) Section of cornea 6 days after infection (control) which represents the peak inflammatory response depicting primarily infiltrating PMN cells. Infiltrating cells were detected as early as 1 day after infection (not shown). (D, E) Section of cornea 6 days after infection stained with the hpa1 antibody. Infiltrating Hpa1-positive cells were detected in the stroma plus positive staining of the corneal epithelium. Similar results were seen with corneas infected 1 day and stained with the Hpa1 antibody (not shown). Magnification: (A, C, E) ×610; (B, D) ×1600.
Figure 3.
 
Immunohistochemistry of the cornea. Uninfected and infected corneas from naïve mice were stained with normal serum, irrelevant IgG (control samples), or Hpa1 antibody, to detect the heparanase protein (days 0, 1, and 6) after infection. (A) Section of normal control cornea not stained with Hpa1 antibody. (B) Uninfected cornea stained with the Hpa1 antibody. Intense staining of the corneal epithelium with faint staining of the stroma is depicted. CE, corneal epithelium; CS, corneal stroma. (C) Section of cornea 6 days after infection (control) which represents the peak inflammatory response depicting primarily infiltrating PMN cells. Infiltrating cells were detected as early as 1 day after infection (not shown). (D, E) Section of cornea 6 days after infection stained with the hpa1 antibody. Infiltrating Hpa1-positive cells were detected in the stroma plus positive staining of the corneal epithelium. Similar results were seen with corneas infected 1 day and stained with the Hpa1 antibody (not shown). Magnification: (A, C, E) ×610; (B, D) ×1600.
Figure 4.
 
Immunohistochemistry of the retina. All sections were stained with either normal serum or irrelevant antibody (control samples) or with the Hpal antibody. All sections represent uninfected eyes, in that there did not appear to be any substantive differences in staining intensity or location of retinal staining for days 0, 1, and 6 after infection. (A) Retinal layers stained with antibody to Hpal. Positive staining was based on orange to brown coloring. OPRL, outer photoreceptor layer; IPRL inner photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL inner plexiform layer; GC, ganglion cells. (B) Hpal-positive retina at a higher magnification. There was intense staining of the IPRL and faint staining of the OPRL and ONL. Positive staining of blood vessels in the OPL was also apparent. (C) Higher magnification showing the intense staining of the IPRL with cytoplasmic staining of the ONL. (D) Positive staining of the cytoplasm of the GCs. Magnification: (A) ×150; (B) ×300; (C) ×610; (D) ×1600.
Figure 4.
 
Immunohistochemistry of the retina. All sections were stained with either normal serum or irrelevant antibody (control samples) or with the Hpal antibody. All sections represent uninfected eyes, in that there did not appear to be any substantive differences in staining intensity or location of retinal staining for days 0, 1, and 6 after infection. (A) Retinal layers stained with antibody to Hpal. Positive staining was based on orange to brown coloring. OPRL, outer photoreceptor layer; IPRL inner photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL inner plexiform layer; GC, ganglion cells. (B) Hpal-positive retina at a higher magnification. There was intense staining of the IPRL and faint staining of the OPRL and ONL. Positive staining of blood vessels in the OPL was also apparent. (C) Higher magnification showing the intense staining of the IPRL with cytoplasmic staining of the ONL. (D) Positive staining of the cytoplasm of the GCs. Magnification: (A) ×150; (B) ×300; (C) ×610; (D) ×1600.
The authors thank Jose Rafols and William Crossland for advice, Malkhan Katar and Julianna M. Berk for technical assistance, and ImClone Systems for their generous gift of the heparanase antibody. 
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Figure 1.
 
Semiquantitative RT-PCR determination of heparanase mRNA expression in the mouse corneas before and during infection with P. aeruginosa. Naïve mice were infected with P. aeruginosa and total RNA samples were prepared from the pooled corneas harvested over an 8-day period. Equal amounts of total RNA (500 ng) from individual samples were used for RT-PCR. Specific primers for mouse heparanase were used to amplify the target genes that were stained by ethidium bromide as bright bands in the dark background. The GAPDH gene was also amplified and used as an internal control.
Figure 1.
 
Semiquantitative RT-PCR determination of heparanase mRNA expression in the mouse corneas before and during infection with P. aeruginosa. Naïve mice were infected with P. aeruginosa and total RNA samples were prepared from the pooled corneas harvested over an 8-day period. Equal amounts of total RNA (500 ng) from individual samples were used for RT-PCR. Specific primers for mouse heparanase were used to amplify the target genes that were stained by ethidium bromide as bright bands in the dark background. The GAPDH gene was also amplified and used as an internal control.
Figure 2.
 
Expression of heparanase protein in the mouse corneas before and during infection as determined by immunoblot analysis. Naïve and immunized mice were infected with P. aeruginosa, and the eyes were harvested over an 8-day infection period. Equal amounts of total protein (12 μg) of each time-point sample were loaded for electrophoresis. A rabbit polyclonal antibody raised to recombinant heparanase fractions was used for immunoblot analysis. MW, molecular weight; r-Hp, recombinant heparanase.
Figure 2.
 
Expression of heparanase protein in the mouse corneas before and during infection as determined by immunoblot analysis. Naïve and immunized mice were infected with P. aeruginosa, and the eyes were harvested over an 8-day infection period. Equal amounts of total protein (12 μg) of each time-point sample were loaded for electrophoresis. A rabbit polyclonal antibody raised to recombinant heparanase fractions was used for immunoblot analysis. MW, molecular weight; r-Hp, recombinant heparanase.
Figure 3.
 
Immunohistochemistry of the cornea. Uninfected and infected corneas from naïve mice were stained with normal serum, irrelevant IgG (control samples), or Hpa1 antibody, to detect the heparanase protein (days 0, 1, and 6) after infection. (A) Section of normal control cornea not stained with Hpa1 antibody. (B) Uninfected cornea stained with the Hpa1 antibody. Intense staining of the corneal epithelium with faint staining of the stroma is depicted. CE, corneal epithelium; CS, corneal stroma. (C) Section of cornea 6 days after infection (control) which represents the peak inflammatory response depicting primarily infiltrating PMN cells. Infiltrating cells were detected as early as 1 day after infection (not shown). (D, E) Section of cornea 6 days after infection stained with the hpa1 antibody. Infiltrating Hpa1-positive cells were detected in the stroma plus positive staining of the corneal epithelium. Similar results were seen with corneas infected 1 day and stained with the Hpa1 antibody (not shown). Magnification: (A, C, E) ×610; (B, D) ×1600.
Figure 3.
 
Immunohistochemistry of the cornea. Uninfected and infected corneas from naïve mice were stained with normal serum, irrelevant IgG (control samples), or Hpa1 antibody, to detect the heparanase protein (days 0, 1, and 6) after infection. (A) Section of normal control cornea not stained with Hpa1 antibody. (B) Uninfected cornea stained with the Hpa1 antibody. Intense staining of the corneal epithelium with faint staining of the stroma is depicted. CE, corneal epithelium; CS, corneal stroma. (C) Section of cornea 6 days after infection (control) which represents the peak inflammatory response depicting primarily infiltrating PMN cells. Infiltrating cells were detected as early as 1 day after infection (not shown). (D, E) Section of cornea 6 days after infection stained with the hpa1 antibody. Infiltrating Hpa1-positive cells were detected in the stroma plus positive staining of the corneal epithelium. Similar results were seen with corneas infected 1 day and stained with the Hpa1 antibody (not shown). Magnification: (A, C, E) ×610; (B, D) ×1600.
Figure 4.
 
Immunohistochemistry of the retina. All sections were stained with either normal serum or irrelevant antibody (control samples) or with the Hpal antibody. All sections represent uninfected eyes, in that there did not appear to be any substantive differences in staining intensity or location of retinal staining for days 0, 1, and 6 after infection. (A) Retinal layers stained with antibody to Hpal. Positive staining was based on orange to brown coloring. OPRL, outer photoreceptor layer; IPRL inner photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL inner plexiform layer; GC, ganglion cells. (B) Hpal-positive retina at a higher magnification. There was intense staining of the IPRL and faint staining of the OPRL and ONL. Positive staining of blood vessels in the OPL was also apparent. (C) Higher magnification showing the intense staining of the IPRL with cytoplasmic staining of the ONL. (D) Positive staining of the cytoplasm of the GCs. Magnification: (A) ×150; (B) ×300; (C) ×610; (D) ×1600.
Figure 4.
 
Immunohistochemistry of the retina. All sections were stained with either normal serum or irrelevant antibody (control samples) or with the Hpal antibody. All sections represent uninfected eyes, in that there did not appear to be any substantive differences in staining intensity or location of retinal staining for days 0, 1, and 6 after infection. (A) Retinal layers stained with antibody to Hpal. Positive staining was based on orange to brown coloring. OPRL, outer photoreceptor layer; IPRL inner photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL inner plexiform layer; GC, ganglion cells. (B) Hpal-positive retina at a higher magnification. There was intense staining of the IPRL and faint staining of the OPRL and ONL. Positive staining of blood vessels in the OPL was also apparent. (C) Higher magnification showing the intense staining of the IPRL with cytoplasmic staining of the ONL. (D) Positive staining of the cytoplasm of the GCs. Magnification: (A) ×150; (B) ×300; (C) ×610; (D) ×1600.
Copyright 2004 The Association for Research in Vision and Ophthalmology, Inc.
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