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Volume 52 (5): 671-682, 2004
Copyright ©The Histochemical Society, Inc.
Immunohistochemical Assessment of Fractalkine, Inflammatory Cells, and Human Herpesvirus 7 in Human Salivary Glands
Center for Oral Biology (LRL,MAF,DJC), Center for Aging and Developmental Biology (HAG), Center for Vaccine Biology and Immunology (SD), and the Departments of Pharmacology and Physiology (DJC), Neurology (HAG), and Microbiology and Immunology (SD), University of Rochester Medical Center, Rochester, New York
Correspondence to: Dr. David J. Culp, Center for Oral Biology, 601 Elmwood Avenue, Box 611, Rochester, NY 14642-8611. E-mail: david_culp{at}urmc.rochester.edu
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Human fractalkine (CX3CL1), a
-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry. (J Histochem Cytochem 52:671–681, 2004)
Key Words: chemokine • infection • parotid • submandibular • labial
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Human fractalkine (CX3CL1), a member of the
-chemokine subfamily, is a 397-amino-acid protein containing a transmembrane domain, an extracellular extended mucin-like stalk, and an adjacent chemokine domain with a unique Cys-X-X-X-Cys motif (Bazan et al. 1997
; Pan et al. 1997). Fractalkine has been shown to mediate cell–cell adhesion through interaction with cells that express the fractalkine receptor CX3CR1 (Bazan et al. 1997; Imai et al. 1997). Moreover, a soluble form of fractalkine is released from the cell surface by proteolytic cleavage of a conserved dibasic motif (Thr-Arg-Arg-Gln) at an extracellular site near the plasma membrane. Soluble fractalkine can function as a chemotactic factor for monocytes, NK-cells, and T-cells expressing CX3CR1 (Bazan et al. 1997; Chapman et al. 2000). Current evidence indicates that fractalkine is expressed constitutively in neurons within specific regions of the brain and in epithelial cells in epidermal tissues and the intestine (Lucas et al. 2001; Hughes et al. 2002; Sugaya et al. 2003). Fractalkine expression is upregulated by endothelial and epithelial cells in epidermal, renal, bronchial, intestinal, and hepatic tissues in response to disease, inflammation, or application of inflammatory mediators (Harrison et al. 1999; Fujimoto et al. 2001; Lucas et al. 2001; Cockwell et al. 2002; Efsen et al. 2002; Hughes et al. 2002; Sugaya et al. 2003). In addition to functioning in cell–cell adhesion and inflammatory cell migration, fractalkine is implicated in cell proliferation (Efsen et al. 2002), cell survival (Meucci et al. 2000; Brand et al. 2002) and in binding to glycosaminoglycans via a heparan sulfate-binding domain (Lortat–Jacob et al. 2002). The importance of fractalkine in modulating tissue inflammatory and immune responses is manifested by the mimicry of this chemokine by the G-glycoprotein of respiratory syncytial virus (RSV), presumably to promote RSV infection of cells that express CX3CR1in the lower respiratory tract (Tripp et al. 2001).
As in other tissues, salivary glands are afflicted by chronic inflammatory and autoimmune disorders and are also targets for viral infection (Rice 1999). Salivary glands are a collection of distinct glandular structures, each of which contributes its own exocrine and fluid secretory constituents into the oral cavity. Three "major" pairs of salivary glands (parotid, submandibular, and sublingual) are linked to the oral cavity through relatively long excretory ducts. In addition, localized just under the oral epithelium are many smaller "minor" glands, named according to their anatomic locations (i.e., labial, lingual, palatal, buccal, and minor sublingual). Salivary glands differ not only in their anatomic location but also in their constituent acinar cell types. The acinar cells of parotid glands are of the serous phenotype (Pinkstaff 1980). Submandibular glands also contain serous acini but in addition have secretory endpieces composed of short tubules of columnar mucous cells capped by serous-like cells referred to as serous demilune cells (Pinkstaff 1980). Labial glands, on the other hand, contain predominantly mucous tubules capped with serous demilune cells (Hand et al. 1999). Surrounding the secretory endpieces of salivary glands are myoepithelial cells, which have branching processes containing actin filaments (Hand et al. 1999). This cell type is present in various amounts among the different salivary glands but tends to be more prevalent in glands that contain a higher proportion of mucous cells (Hand et al. 1999).
Salivary glands are a major site for a persistent and productive infection by human herpesvirus (HHV)-7 (Sada et al. 1996). HHV-7 infection occurs primarily in early childhood (Wyatt et al. 1991; Clark et al. 1993), and infectious virus is readily isolated from saliva (Hidaka et al. 1993). Nevertheless, the pattern and cellular localization of HHV-7-infected cells in the two largest major salivary glands, the parotid and submandibular glands, has yet to be elucidated. Moreover, mechanisms by which HHV-7 gains entry to salivary gland cells and evades host immune responses are largely unknown. However, HHV-7 encodes two proteins, U12 and U51, with homology to a seven-transmembrane protein encoded by the US28 gene of human cytomegalovirus (HCMV) (Rosenkilde et al. 2001). The US28 gene product appears to mimic CX3CR1 in its binding to fractalkine and enhances virion–cell fusion in a cell type-specific fashion (Pleskoff et al. 1998; Fraile–Ramos et al. 2001). In a similar manner, HHV-7 U12 and/or U51 may interact with salivary fractalkine to enhance viral entry or spread (Fraile–Ramos et al. 2001). One or both of these proteins may also modulate chemokine signaling pathways and subsequent immune responses by sequestering soluble tissue fractalkine (Fraile–Ramos et al. 2001).
It is unknown whether fractalkine is expressed in human salivary glands. Therefore, the first aim of this study was to evaluate fractalkine protein expression and its cellular localization by immunocytochemistry (ICC) in three different human salivary glands: parotid, submandibular, and labial. All tissue specimens were normal in appearance when initially excised. Nevertheless, fractalkine expression was found to be highly variable, with some specimens expressing undetectable or very low levels of fractalkine. Given such variable expression levels of fractalkine, we further explored whether inflammatory cell infiltration or HHV-7 infection of salivary cells was related to the expression of fractalkine. We reasoned that if fractalkine is required for either process, then inflammatory cells and/or HHV-7-infected cells will be associated only with epithelial cells that express fractalkine. Our results are consistent with a role for fractalkine in the infiltration of inflammatory cells, whereas regions in which cells are infected by HHV-7 vary in fractalkine expression.
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Materials and Methods |
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All procedures for acquiring human tissues were approved by the Research Subjects Review Board of the University of Rochester. Sections of labial glands were obtained from the Department of Pathology, University of Rochester School of Medicine and Dentistry, Rochester, NY. Labial glands were obtained at biopsy for histological diagnosis of Sjögren's syndrome and were classified clinically as negative. Submandibular and parotid gland tissues were obtained either through the Cooperative Human Tissue Network (Ohio State University in Columbus, OH and University of Pennsylvania Medical Center in Philadelphia, PA) or from patients undergoing oral surgery locally. Normal-appearing parotid and submandibular gland tissues (other than specimens #8 and #9; see Table 1) were obtained during surgery for glandular carcinoma except for specimens #12 and #13, in which a schwannoma and a melanoma, respectively, was juxtaposed to glandular tissue. Specimens #8 and #9 were removed because of chronic sialoadenitis. In all cases, excised tissues were immediately formalin-fixed, embedded in paraffin, and sectioned (5 µm). In almost all cases, paraffin blocks contained two tissue samples and serial sections were mounted two per slide. Each experimental condition, once optimized, was performed at least twice on slides from each specimen.
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Immunohistochemical (IHC) detection of fractalkine or HHV-7 was performed as described previously, with minor modifications (Fallon et al. 2003
). To unmask antigenic sites in formalin-fixed tissues, we first incubated sections for 10 min in 5% urea at 95C. After inhibition of endogenous peroxidase activity (100% methanol containing 0.3% H2O2) and incubation with 10% normal horse serum (NHS; Vector Laboratories, Burlingame, CA) in PBS, sections were incubated overnight (4C) with primary antibody or an appropriate negative control (diluted in 10% NHS in PBS). Primary antibody to human fractalkine (2 µg/ml, goat anti-human fractalkine; R&D Systems, Minneapolis, MN) was prepared using recombinant human fractalkine chemokine domain as immunogen, with subsequent purification of fractalkine-specific IgG by antigen-affinity chromatography. Antibody to HHV-7 (14.5 µg/ml, IgG purified from mouse monoclonal 5E1; Advanced Biotechnologies, Columbia, MD) was shown to be reactive against the tegument protein pp85 of HHV-7 (Kempf et al. 1998). Negative controls included preabsorption with a fivefold excess of human fractalkine chemokine domain peptide, amino acids 25 – 100 (R&D Systems), normal goat IgG (2 µg/ml; R&D Systems), and normal mouse IgG (14.5 µg/ml; Sigma, St Louis, MO). Sections were washed (PBS + 0.05% Tween-20) and incubated for 1 hr with either biotinylated horse anti-goat or biotinylated horse anti-mouse/rabbit (both from Vector) diluted to 10 µg/ml in PBS. Immunodetection was performed using the avidin–biotin–peroxidase complex method with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as peroxidase substrate and Ni enhancement (Vectastain Elite kit; Vector). For dual detection of fractalkine and either CD3 or CD68, sections were again blocked (30 min in 10% NHS in PBS) followed by 1-hr incubation at room temperature with rabbit anti-human CD3 or mouse monoclonal anti-human CD68 (both from DAKO; Carpinteria, CA). Anti-human CD3 and its negative control were applied as received from DAKO without dilution. Anti-human CD68 and normal mouse IgG (negative control) were diluted to 3.8 µg/ml in 10% NHS in PBS. Sections were treated with secondary antibody (biotinylated horse anti-mouse/rabbit) as described above and immunodetection performed using the avidin–biotin–alkaline phosphatase complex method (Vectastain ABC-AP kit and Vector Red substrate; Vector) according to the manufacturer's directions. As positive controls in dual staining, we used sections of human tonsil (DAKO) or lymphoid tissue juxtaposed to portions of a human parotid gland obtained locally during surgery. Because antibody titers were not sufficient to allow dual staining for fractalkine and HHV-7, serial sections were examined.
After antibody detection, sections were washed, dehydrated, cleared in xylene, and mounted in Refrax (Anatech; Battle Creek, MI). Sections were examined with a Nikon Eclipse E800 microscope (Nikon; Melville, NY) under DIC (differential interference contrast) optics. Digital images were captured with a Spot 2 digital camera and software (Diagnostic Instruments; Sterling Heights, MI) and prepared for figures using Adobe Photoshop software (Adobe Systems; San Jose, CA).
Sections from each tissue sample were processed simultaneously with primary antibody and appropriate negative control as one of two groups: (a) sections from all labial gland samples, or (b) sections from all samples of parotid and submandibular glands. Stained sections were examined under low-power (x100) light microscopy and scored qualitatively by a single observer (DJC) using a four-plus system. After examination of all samples and negative controls for a given antigen, the sample with the most abundant cellular staining throughout the tissue, regardless of cell type, was considered to represent 100% maximal staining. This specimen and all others judged to have total staining >75% to 100% of maximal were given a score of four-plus. Specimens judged to have total staining >50% to 75% of maximal were given a score of three-plus. Similarly, specimens with total staining of >25% to 50% and >0% to 25% of maximal were given scores of two-plus and one-plus, respectively. No obvious differences in the distribution of cell types were noted among the specimens. Scoring is therefore with respect to all glandular samples for each antigen and is not a comparison among antigens.
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Detection of fractalkine in ductal and acinar cells was highly variable among the 18 specimens of salivary glands (Table 1; Figure 1) . In one specimen, #11, fractalkine expression was not detected in all three sections examined (not shown; Table 1). The greatest amount of immunostaining was observed in submandibular specimens #9 and #12. These two specimens were therefore scored four-plus (Table 1; see Materials and Methods for scoring details). The nuclear region of almost all epithelial cells in both specimens stained intensely (Figure 1A). Furthermore, dark cytoplasmic staining and reactivity along the luminal membrane was readily observed in columnar duct cells (Figures 1A and 2C) . Occasionally, immunoreactivity in these two submandibular gland specimens was observed along the lateral and/or luminal membrane of mucous cells (Figure 2F) and serous acinar cells (not shown). The cytoplasm of mucous cells (restricted to basal regions), serous acinar cells, serous demilune cells, and even myoepithelial cells was moderately stained (Figures 1A and 2G). In specimens with lower levels of immunostaining, general trends were noted. First, in samples with the lowest immunoreactivity (one-plus scoring; Table 1), staining was predominantly localized to columnar duct cells (Figure 1D). Specimens with two-plus and three-plus scoring (Figures 1B and 1C) were characterized by immunostaining of the great majority of columnar ducts. Higher scoring of these samples was due primarily to immunoreactivity of increasing proportions of acinar cells.
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Interestingly, the dark cytoplasmic staining observed in serous acinar cells and serous demilune cells of submandibular gland specimens #9 and #12 was not seen in the analogous cell types of the parotid and labial gland specimens, respectively (Figures 1B and 1C). In all three types of salivary glands, we found examples of luminal membrane staining in columnar duct cells and cuboidal intercalated duct cells (Figure 2D). As described above for serous cells in submandibular glands, serous cells in parotid glands also displayed occasional luminal membrane staining (Figure 2E). Immunoreactivity in mucous cells of labial glands was similar to that observed in mucous cells of submandibular glands. Immunostaining was more pronounced in the perinuclear region, with less and more diffuse staining of the basal cytoplasm, away from densely packed mucous granules (Figure 1C) and occasionally along luminal plasma membranes (not shown). Fractalkine expression was also found in endothelial cells of vascular structures in tissue samples (Figures 2A and 2B), usually in regions containing strong staining by surrounding epithelial cells. Staining was absent in control sections that were processed with either normal goat IgG (Figure 1E) or with antibody preabsorbed with the antigen peptide (not shown).
Large clusters of CD3-positive cells were detected only in specimens #8 and #9. These clusters were localized in regions of loose connective tissue that contained vascular structures and large duct structures (Figure 3A) . Adjacent duct structures and nearby acini were consistently positive for fractalkine. In some sections there was an obvious gradient in the intensity of epithelial cell fractalkine staining; with less intense staining the further removed epithelial cells were from a locus of CD3-positive cells (Figure 3A). Some CD3-positive cells were also positive for fractalkine (Figure 3C). In sections from specimen #2, we found only a single small cluster of about five cells that were CD3-positive (not shown). All other specimens displayed negative staining, even in the presence of high levels of fractalkine expression (e.g., specimens #7, #12, and #13; see Table 1). Cells positive for CD68 were present primarily as only one (specimen #9) or two (specimen #8) small groups of about a dozen cells within the loose connective tissue near large duct structures (Figure 3D; Table 1). A subset of CD68-positive cells was also positive for fractalkine. Based on alternate staining of serial sections, these groups of CD68-positive cells were mostly localized to regions independently of CD3-positive cells (not shown). On the other hand, approximately one-quarter of the clusters of CD3-positive cells in specimen #8 contained one or two cells positive for CD68.
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Positive immunostaining for HHV-7 was present in sections from all but four specimens (Table 1). Overall, the proportion of cells that stained positive for HHV-7 was much less than that for fractalkine. For example, in tissue sections from specimen #12, which displayed the most immunoreactivity for HHV-7, only about half of the duct and acinar cells were positive. As described above for fractalkine, specimens with the lowest HHV-7 immunoreactivity (one-plus scoring; Table 1) were stained mostly in one or a few groups of columnar duct cells. Duct cell staining in all three glands was characterized by diffuse cytoplasmic and/or perinuclear immunoreactivity (Figure 4A) . In some cases, staining of the luminal membrane was also seen (Figure 4A). Specimens with a larger distribution of stained cells (and hence increased scoring) displayed increasing proportions of acinar cell immunoreactivity. Positive immunoreactivity in serous acinar cells of parotid and submandibular glands ranged from light to very dark diffuse cytoplasmic staining, even within the same or adjoining acinus (Figure 4B). In some cases cytoplasmic staining was of a more granular appearance (Figure 4C). A more punctate cytoplasmic staining was more apparent in serous demilune cells of submandibular and labial specimens (Figure 4D). The degree of staining in duct cells was generally similar to that shown in Figure 4A, and was less intense as seen in most serous and/or serous demilune cells. Few mucous cells displayed immunoreactivity for HHV-7. When present, mucous cell staining was diffuse and was localized to the basal and perinuclear cytoplasm and/or near plasma membranes (Figure 4D). In addition, we found inconsistent results in serial sections probed alternately for the cellular localization of HHV-7 and fractalkine. For example, groups of cells that stained intensely for fractalkine were either negative for HHV-7 (not shown) or displayed strong HHV-7 immunoreactivity (Figures 4F and 4G). Conversely, we found cells positive for HHV-7 that did not stain for fractalkine (Figures 4H and 4I).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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On the basis of its presence in epithelial components within normal tissues from epidermis, tonsils, and colon, it has been proposed that fractalkine is constitutively expressed in epithelial cells (Lucas et al. 2001
). In contrast, fractalkine is reportedly absent in normal bronchial epithelium (Fujimoto et al. 2001) or is expressed in very low levels in normal tissues from kidney (Cockwell et al. 2002) and liver (Efsen et al. 2002). We found highly variable expression of this chemokine in specimens of normal tissues from three different salivary glands, suggesting that fractalkine is not expressed constitutively by salivary epithelial cells. The varied anti-fractalkine immunoreactivity observed in salivary tissues, even within the same specimen, may be related to localized signals that induce or even downregulate fractalkine expression. In aortic smooth muscle cells, fractalkine was shown to be upregulated via nuclear factor 
B-dependent signaling (Chandrasekar et al. 2003). Induction of fractalkine can occur either in an autocrine manner via activation of CX3CR1 or in response to interleukin-1 (Muehlhoefer et al. 2000) or to tumor necrosis factor-
(Harrison et al. 1999; Lucas et al. 2001; Chandrasekar et al. 2003; Sugaya et al. 2003). In addition, autocrine signaling by fractalkine in intestinal epithelial cells mediates activation of extracellular signal-regulated kinase as well as the release of interleukin 8 (Brand et al. 2002). Fractalkine may therefore modulate both cell survival signals and local immune responses. Future elucidation of extra- and intracellular signals controlling fractalkine expression by salivary epithelial cells will likely lead to identification of important autocrine or paracrine mechanisms that regulate cell physiology in salivary glands.
Fractalkine is considered to function as an integral type I plasma membrane protein, either as the intact molecule or as the soluble extracellular fragment after proteolytic cleavage (Chapman et al. 2000; Lucas et al. 2001). In the present study, fractalkine was indeed localized to plasma membranes of salivary epithelial cells. Because of the antigen retrieval step and the extensive washings used in the processing of tissue sections, it is unlikely that soluble fractalkine remained for subsequent detection in our specimens. On the other hand, we found predominant fractalkine staining within the nuclear region of cells. This staining pattern is not an artifact contributed by the antigen retrieval method or by the secondary staining protocols, as evidenced by the fact that the same procedures and reagents were used for determining HHV-7 immunolocalization in adjacent sections. Furthermore, staining in the nuclear region was observed in the absence of the antigen retrieval step and/or when alternate secondary antibody and detection systems were used, although staining intensities were markedly reduced (not shown). To date, the immunoreactivity of fractalkine in other cell types has not been localized exclusively to plasma membranes. Immunostaining has been reported in the cytoplasm of keratinocytes (Sugaya et al. 2003) and in punctate vesicle-like structures adjacent to nuclei of neuron cell bodies (Tong et al. 2000). The staining observed in the nuclear region of salivary cells may represent newly-synthesized fractalkine in the rough endoplasmic reticulum. Subsequent steps in the transport and/or glycosylation of fractalkine may mask the chemokine antigenic site. The relatively lower levels of membrane staining might be explained by the rapid proteolytic cleavage of fractalkine after insertion in the plasma membrane. Alternatively, the observed immunoreactivity in the nuclear region may represent, in part, fractalkine chemokine domains in the nuclei of salivary cells or in specialized endocytic vesicles. Elucidation of the life cycle of fractalkine (i.e., synthesis, intracellular processing and transport, membrane insertion, proteolytic cleavage, or possible endocytosis) is beyond the scope of the present study and requires the use of model systems other than paraffin sections of pathological specimens.
A number of studies have demonstrated a role for fractalkine in tissue responses to injury and subsequent inflammation. For example, soluble fractalkine released from the cell surface is a chemotactic factor for monocytes, NK-cells, and T-cells expressing CX3CR1 (Bazan et al. 1997; Chapman et al. 2000). Furthermore, membrane-bound fractalkine may assist in adhesion of CX3CR1-expressing leukocytes to epithelial cells and in retention of leukocytes in the surrounding connective tissue (Imai et al. 1997; Haskell et al. 1999; Chakravorty et al. 2002). As a case in point, during renal inflammation subsets of leukocytes (CD68-positive macrophages and CD3-positive T-cells) were found adjacent to fractalkine-expressing tubule epithelial cells (Cockwell et al. 2002). In the present study we found large clusters of CD3-positive cells as well as cells positive for CD68 only in tissue sections from two submandibular glands (specimens #8 and #9). Both glands were removed because of chronic sialoadenitis, whereas all other submandibular and parotid specimens were from tissue samples removed due to either intra- or extraglandular carcinomas. Therefore, the presence of inflammatory cells in specimens #8 and #9 is not surprising, although the presence of a subset of leukocytes expressing fractalkine should be noted. The expression of fractalkine by tissue macrophages has been reported previously (Fraticelli et al. 2001; Greaves et al. 2001). Furthermore, primary macrophages upregulate fractalkine in response to interleukin-4, suggesting that fractalkine expression may serve as a marker for T-helper type 2 immune responses (Greaves et al. 2001). These results are consistent with the presence of infiltrating lymphocytes expressing type 2 cytokines in labial glands of patients with Sjögren's syndrome (Mitsias et al. 2002).
Leukocytes identified in specimens #8 and #9 were consistently found adjacent to epithelial cells that were intensely positive for fractalkine. In some cases fractalkine staining was gradually less intense in epithelial cells that were further removed from a cluster of CD3-positive cells. These results are consistent with a function of fractalkine in the recruitment and/or retention of inflammatory cells in salivary glands. On the other hand, the absence of detectable inflammatory cells in other specimens in which high levels of fractalkine was expressed indicates that other factors are required for the recruitment of leukocytes into salivary glands. One such factor may be the release from epithelial membranes of soluble fractalkine, the more chemoattractant form of the chemokine. As mentioned above, it is likely that soluble fractalkine was washed out of our specimens during processing.
Although HHV-7 is a persistent and productive infectious agent in human salivary glands, the cellular distribution of virions has been described only for labial minor glands (Yadav et al. 1997). Duct cells were consistently labeled in all 20 specimens examined, whereas staining in serous demilune cells and mucous cells was limited to scattered foci of cells (Yadav et al. 1997). In the present study we did not observe HHV-7 immunoreactivity in all labial specimens, nor were duct cells consistently stained in infected glands. These differences may be related to the antibody probes used. In the previous study, monoclonal antibody KR4 was used to detect viral antigen (Yadav et al. 1997). To our knowledge, neither the specificity of this antibody nor its target epitope has been described. We used monoclonal antibody 5E1, which is directed against tegument protein pp85 of HHV-7 (Stefan et al. 1997; Kempf et al. 1998). This antibody was previously listed as detecting virus in six of ten salivary glands of unspecified type (Kempf et al. 1998). In a separate study, positive staining of parotid acinar cells served only as a positive control experiment (Kempf et al. 1997). Nevertheless, neither study provided a complete account of the cellular localization of 5E1 immunoreactivity in parotid glands or other salivary glands. In general, we found that 5E1 immunoreactivity in all three types of salivary glands was limited to foci of cells, and that serous and serous demilune cells were more intensely stained than duct or mucous cells. The higher intensity of staining in serous and demilune cells may represent either a greater production of virions by these two cell types and/or the cytoplasmic accumulation of tegument-containing material at a biosynthetic site or depot (Stefan et al. 1997). Alternatively, virus may be produced no more efficiently but is released at a slower rate and therefore stored in these two cell types. All epithelial cell types in salivary glands are therefore targets for HHV-7 infection, but the ability of each cell type to support the synthesis, assembly, and release of viral particles may be strikingly different. In addition, unlike labial and parotid glands, all of our submandibular specimens were positive for 5E1 immunoreactivity and with scores of at least two-plus. Although our sampling of glandular tissues is not statistically relevant, it is worth noting that these results are consistent with those of Sada et al. (1996), in which HHV-7 DNA was detected by PCR in all 59 specimens of submandibular glands tested, compared with 85% and 59% for parotid and labial glands, respectively. Submandibular glands may therefore be more susceptible to infection by HHV-7 compared with other salivary glands.
Despite the presence of foci of cells infected with HHV-7 in the majority of specimens examined, inflammatory cells were detected in only three cases. These results are consistent with the evolution by HHV-7 of mechanisms to target salivary epithelial cells for reproduction and to evade host defenses, leading to the persistent release of infectious virus into saliva (Wyatt and Frenkel 1992; Clark et al. 1993). We speculated that the putative fractalkine-binding proteins encoded by HHV-7 genes U12 and U51 may assist in viral entry by binding to cell surface fractalkine of epithelial cells. Alternatively, if fractalkine is induced by viral infection, then one or both these proteins could conceivably aid in evasion of host defenses by sequestering extracellular fractalkine, thus attenuating the recruitment of CX3CR1-expressing cells to the site of infection (Tortorella et al. 2000). Another scenario is for HHV-7 entry to promote the downregulation of constitutive fractalkine expression (Tortorella et al. 2000). Our results are inconsistent with all of these putative relationships between HHV-7 infection and fractalkine expression. For example, we observed cases in which cells infected with HHV-7 were either positive or negative for fractalkine, suggesting that infection does not necessarily lead to up- or downregulation of fractalkine expression. Furthermore, because fractalkine expression is not constitutive and its expression by infected cells is variable, fractalkine is probably not an absolute requirement for viral entry. We recognize that a functional interrelationship between cellular fractalkine and HHV-7 may still exist in salivary gland infection and/or immune evasion, but in a more complex manner. Given the absence of an in vitro model system in which to study the interactions between salivary epithelial cells and HHV-7, our results nevertheless provide initial insights into the cellular distribution of a viral parasite in salivary glands. In addition, we describe for the first time the expression in salivary glands of a unique protein with chemokine activity that is likely to participate in other cellular functions that have yet to be elucidated.
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Acknowledgments |
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Supported by grant RO1 DE14194 and by a fellowship (HL07126) to MAF from the National Institutes of Health.
We wish to thank Ashley John Grillo and Sally Chuang for excellent technical assistance, and Dr Seth Perry for valuable discussions. We also thank Dr Gene Watson for assistance in procuring tissue samples.
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Footnotes |
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Received for publication October 30, 2003; accepted January 14, 2004
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