Acute-phase proteins (APP) are serum proteins that are upregulated and downregulated following homeostasis disturbance such as infection, inflammation, tissue injury, or neoplasia 1. The synthesis of APP such as serum amyloid A and C-reactive protein mainly occurs in the liver and is induced by proinflammatory cytokines such as interleukin 6 and tumor necrosis factor 2. In pigs, studies on extrahepatic production of APP have been focused on the respiratory tract. An extrahepatic production of C-reactive protein has been reported in vascular smooth muscle cells 3, pulmonary fibroblasts, and endothelial cells in the lung 4. Moreover, haptoglobin (Hp) has been located in airway epithelial cells and immigrated leukocytes 2 and in intestinal lymph nodes and peripheral lymphoid tissues of pigs 5. Serum Hp has been used as an inflammatory marker to assess animal health 6, monitor antibiotic therapy, and as a tool for veterinary inspection at slaughter 7. Measurements of Hp in saliva samples have been proposed as suitable alternatives to serum as they have several analytical advantages 8. Saliva is obtained using a simple noninvasive and minimally stressful method that can be performed by personnel with minimal training.
Aim of the work
The present study aimed to investigate the possible extrahepatic localization of Hp in the submandibular gland.
Materials and methods
Twenty adult cows were obtained from Benha abattoir, Benha city, Qaliobia province, Egypt, and were used throughout the experiment. The cows were given a balanced diet with free access to water. All animal procedures were performed according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals. Animals were divided into two groups: group I (10 cows) was considered as the control group (the healthy group); group II (10 cows) consisted of diseased animals.
Diseased animals were randomly chosen among cows belonging to the same abattoir with clinical signs of disease and poor body condition that rendered them unsuitable for slaughter. Thereafter, the animals were killed by cutting their throat and complete postmortem examinations were performed.
Samples from the submandibular gland from both healthy and sick animals were collected in 10% neutral buffered formalin and processed for paraffin blocks. Sections of 5 μm thickness were cut and stained with H&E. All sick animals presented gross and microscopic lesions compatible with porcine respiratory disease complex.
Liver samples were collected as positive control to observe Hp.
Liver and submandibular glands from healthy and diseased animals were processed for immunohistochemical analysis using the same protocol based on the avidin–biotin peroxidase complex technique (ABC) as previously described 9. Briefly, tissue sections were dewaxed and rehydrated through graded ethanol and the endogenous peroxidase activity was quenched in H2O2 3% in methanol for 30 min. The sections were washed with PBS and incubated for 30 min at room temperature with 100 μl per slide of blocking solution (10% normal goat serum; Sigma Chemical Company, St Louis, Missouri, USA) in a humid chamber. For antigen retrieval, tissues were placed in citrate buffer (10 mmol/l, pH=6) and heated for 5 min at 450 W followed by 6 min at 150 W in a microwave. Tissue sections were incubated overnight with anti-porcine Hp (100 μl of 1:50 dilution from an aliquot of 0.6 mg/ml) at 4°C in a humid chamber followed by30 min incubation at room temperature with a secondary antibody (biotinylated polyclonal goat anti-mouse immunoglobulin; Dako Denmark A/S, Glostrup, Denmark) (100 μl of 1:50 dilution from an aliquot of 0.8 mg/ml). An avidin–peroxidase complex (Vector Laboratories, Burlingame, California, USA) was applied for 1 h at room temperature. Labeling of porcine Hp in tissue sections was visualized or revealed using a specific substrate according to the manufacturer’s instructions (NovaRED substrate kit; Vector Laboratories). Finally, sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. For negative controls, tissue sections were analyzed as reported above, but by replacing the primary antibody with blocking solution (primary antibody-omitted negative control).
The submandibular gland of the control group showed mixed serous and mucous acini arranged in lobules, as well as striated ducts and interlobular ducts (Figs. 1 and 2).
Examination of the liver from diseased animals revealed positive Hp, which was present as perinuclear, small-to-medium-sized globules in the cytoplasm of the hepatocytes (Fig. 3).
In contrast, no immunolabelling of Hp was seen in the liver of healthy animals (Fig. 4). In diseased animals immunolabelling of scattered glandular epithelial cells in glandular acini of the submandibular gland was observed, coinciding with a more intense hepatic presence of Hp. Some of these glandular epithelial cells showed diffuse, cytoplasmic immunostaining (Fig. 5). In addition, the presence of Hp was also observed within the cytoplasm of some epithelial cells and in the ducts of the submandibular gland, showing two patterns of immunolabelling: multiple, immunolabelled globules occupying the whole cytoplasm (Fig. 6); and diffuse, cytoplasmic immunolabelling, which was more intense at the apical border of the cells (Fig. 7).
In the submandibular gland from healthy animals, a mild immunolabelling was observed within the cytoplasm of duct epithelial cells, whereas no glandular epithelial cell was immunolabelled (Fig. 8). In addition, no staining was observed in the negative control (Fig. 9).
In the present study we have used a monoclonal antibody against porcine Hp. By using this antibody we have evaluated the possible local presence of Hp in the submandibular salivary gland of healthy cows in order to gain knowledge about the source of the Hp. When the effective production of Hp was investigated by immunohistochemistry in liver tissue sections, immunostaining was observed in the cytoplasm of hepatocytes. Extracellular staining was also observed in the lumina of blood vessels, showing, possibly, the active synthesis and secretion of Hp in the liver as has been reported elsewhere 1. Previous studies have detected Hp mRNA in liver tissues, being higher in diseased animals 5.
However, to the author’s knowledge, no immunohistochemical analysis has been performed until now on porcine submandibular sections. The results of the present study may confirm that the acute-phase response is produced in the liver involving the hepatic synthesis of Hp, as this protein was observed in the cytoplasm of hepatocytes. In addition, the positive immunostaining of hepatocytes against Hp served as a positive control for Hp production in the animals included in the study.
The presence of Hp in submandibular salivary glands was demonstrated in the present study, an intense production of Hp in sick animals being observed compared with the lack of a marked immunolabelling in control animals. The localization of Hp observed in submandibular salivary glands may be related to an extrahepatic synthesis of this APP in this tissue, which may be supported by the immunostaining of both glandular and ductal epithelial cells of the submandibular salivary gland. However, further studies, including analysis of mRNA, would be necessary to confirm this hypothesis as several mechanisms of transport of proteins from serum into salivary gland ducts have been reported 10–13, which could be responsible for the localization of specific proteins in duct cells. Those studies could provide new insights into the importance of Hp production in local tissues and its implication and role in cases of a systemic disease.
This study has revealed an extrahepatic presence of Hp in submandibular salivary glands of animals through an immunohistochemical analysis using a specific mAb against porcine Hp. Extrahepatic localization, in addition to an increase in the hepatic systemic production of Hp, would explain the increase in Hp levels found in saliva samples in inflammatory conditions.
Conflicts of interest: There is no conflict of interest to declare.
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