Michael J. Day, BSc, BVMS (Hons), PhD, DSc, DECVP, FASM, FRCPath, FRCVS
School of Clinical Veterinary Science, University of Bristol, Langford, United Kingdom
This presentation reviews recent studies from the author's laboratory that characterize the nasal immune system in normal young and adult dogs and defines changes in these immune parameters that occur in idiopathic lymphoplasmacytic rhinitis, sinonasal aspergillosis and nasal carcinoma.
The Normal Canine Nasal Immune System
Recent studies have evaluated aspects of immune defense in the canine nasal mucosa and nasopharynx. Immunohistochemistry has been used to examine the nature and distribution of leucocyte subsets within the nasal mucosa of puppies less than 6 months of age and adult dogs aged more than 1 year. The canine nasal cavity lacks organized mucosal lymphoid aggregates but does have diffuse lymphoid populations. IgA plasma cells predominate over IgM and IgG plasma cells within the nasal mucosa and are particularly distributed around glandular tissue. There are more IgA plasma cells within the nasal mucosa than the lower respiratory tract mucosa and this observation is consistent with a dominance of secretory IgA within the upper respiratory tract. Mast cells are also prominent within the nasal mucosa and are present in greater number than in the lower respiratory tract. These cells localize immediately beneath the mucosal epithelium. Similarly, there are significant numbers of antigen presenting cells (APC) expressing either CD1 (a dendritic cell marker) or class II antigens of the Major Histocompatibility Complex (MHC) within the nasal mucosa, with more MHC class II+ cells at this location than in the lung. Intriguingly, the nasal mucosa of puppies has more mast cells and MHC class II+ APC than that of adult dogs, but there are more CD1+ dendritic cells within the nasal mucosa of adults. The greatest numbers of CD3+ T cells and T cells of the CD8 subset are also found in the nasal mucosa compared with the lower respiratory tract. All types of T-cell (CD3+, CD4+, CD8+ and T cells expressing the αβ T-cell receptor, TCR) are more prominent within the nasal mucosa of adult dogs compared with pups. Of note is the relative absence of T cells expressing the γδ TCR which are, by contrast, enriched in the enterocyte layer of the canine intestine. Taken together, these data suggest that the nasal mucosa has a larger complement of immune cells and that this expands with age. These observations are consistent with the greater exposure of the nasal mucosa to inhaled antigen and with increasing such exposure over time. A similar investigation has characterized the immunological composition of the nasopharyngeal tonsil of the dog. This structure is located in the caudal nasopharynx just distal to the openings of the auditory tubes. By contrast to the nasal cavity, this structure is of maximum size in puppies (mean age 0.3 years) compared with adults (mean age 8.8 years). Within this structure IgA plasma cells dominate over IgG and IgM cells and are greater in number in adults than pups. There are mast cells and APC (including CD1+ dendritic cells) immediately beneath the lining epithelium and these are more numerous in pups than in adults. The lymphoid tissue of this tonsil includes B-cell follicular areas with a surrounding T cell zone which includes both CD4+ (dominant) and CD8+ (fewer) T cells with dominant expression of the αβ TCR. T cells are also found in lower number within the mantle zone and germinal centre of the follicles. The same study investigated seven further areas of the nasopharyngeal mucosa by examination of HE stained sections. In these samples there was minimal diffuse lymphoid tissue characterized by scattered individual lymphocytes and plasma cells and a population of intraepithelial lymphocytes. These were not further characterized by immunohistochemistry. In order to characterize functional aspects of nasal immunity, biopsy samples were collected from normal dogs (four laboratory beagles and nine client-owned dogs that were euthanized for nonrespiratory disease) and subject to real-time reverse transcriptase polymerase chain reaction (RT-PCR) to determine the normal expression of genes encoding cytokines and chemokines. Transcripts from all genes examined (IL-4, IL-5, IL-6, IL-8, IL-12p35, IL-12p40, IL-12p19, IL-13, IL-18, IFN-gamma, TNF-alpha, TGF-beta, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin-2 and eotaxin-3) were detected within the normal nasal mucosa suggesting that this is an immunologically active site.
Idiopathic Lymphoplasmacytic Rhinitis (LPR)
LPR is characterized by the histopathological appearance of inflammation of the nasal mucosa dominated by lymphocytes and plasma cells. Recent studies have failed to provide conclusive evidence that this disease has an underlying infectious cause although it has been suggested that this may represent one form of immune response to inhaled fungal antigen. It is generally considered that LPR may represent a form of allergic upper respiratory tract disease. We have recently applied the same real-time RT-PCR reactions to nasal mucosa biopsies collected from 8 dogs (aged 3 - 14 years) with LPR confirmed by rhinoscopic and histopathological examination and exclusion of other differentials. Relative to controls, biopsies from dogs with LPR revealed selective increases in gene expression for IL-5, IL-8, IL-10, IL-12p40, IL-12p19, IL-18, TNF-α TGF-β and MCP-2 and -3. Elevated IL-5 expression is consistent with a partial Th2 regulated immunity consistent with an allergic pathogenesis. There is also presumptive activity of regulatory T cells that secrete IL-10 and TGF-β. Up-regulation of IL-8 and MCP-2 and -3 is consistent with recruitment of some granulocytes and monocytes and correlates with the minor presence of these inflammatory cell types histologically. The combination of IL-12p40 and p19 suggests the formation of IL-23 (comprised of these two components, whereas IL-12 consists of p35 and p40) and a possible role for Th17 cells.
Sinonasal Aspergillosis (SNA)
SNA is a major infectious disease of the canine sinuses and nasal cavity and is generally caused by infection with Aspergillus fumigatus. In similar studies we have characterized the local nasal immune response to this infection. Histologically, the fungal plaques that are typical of this disease appear to cover the mucosa but fungal elements do not infiltrate into the underlying tissue. The extensive tissue and bone destruction that occurs in this disease appears to be largely mediated by the inflammatory response. This inflammatory response has been characterized immunohistochemically and includes a dominance of IgG plasma cells over IgM and IgA cells (in contrast to the normal balance of nasal immunity), numerous MHC class II+ APCs, early emigrant neutrophils and macrophages expressing L1, and a mixture of CD4+ and CD8+ T cells. The functional nature of this immune response is distinct from that of LPR. Relative to controls, there is up-regulation of gene expression for the following cytokines and chemokines: IL-6, IL-8, IL-10, IL-12p19, IL-12p35, IL-12p40, IL-18, TNF-α TGF-β, IFN-γ, eotaxin-2 and MCP-1, -2, -3 and -4. This suggests a more polarized Th1 immune response in this disease (IL-12, IL-18 and IFN-γ) with a role for Th17 cells and recruitment of neutrophils, eosinophils and monocytes. The immunoregulatory activity (IL-10 and TGF-β) may occur to limit the tissue damage, but concurrently permits persistence of the infection accounting for the chronic nature of this disease.
Our most recent investigation contrasts the nasal immune response in an idiopathic allergic and infectious disease with that occurring during intranasal neoplasia. The local immune response was examined by immunohistochemical assessment of biopsies from 31 dogs with nasal carcinoma, including 12 transitional carcinomas, 11 adenocarcinomas (8 with an acinar pattern and 3 with a tubulopapillary pattern), 3 squamous cell carcinomas, 3 neuroendocrine carcinomas and 2 undifferentiated carcinomas. CD3+ T cells were observed in only 13 of the 31 canine nasal carcinomas. In six cases, numerous CD3+ cells were present while in the 7 other cases, only sparse positive cells were found. These cells were found mainly at the periphery of the tumour with few CD3+ cells infiltrating the tumour tissue. The number of CD3+ cells associated with carcinoma tissue was lower than that found in normal canine nasal mucosa. There were significantly more CD3+ cells associated with transitional carcinoma than with adenocarcinoma. Some canine tumour cells occasionally expressed cytoplasmic MHC II. MHC II+ cells were observed in the sections from 19 canine carcinomas and were mainly found in small clusters at the periphery of the tumour. There were significantly more MHC II+ cells in transitional carcinomas than in adenocarcinomas. L1+ neutrophils and macrophages were the most abundant population of inflammatory cells found in canine nasal carcinomas (in 24 cases). There was no difference in the number of L1+ cells between the different types of carcinomas. IgG+ and IgA+ cells were found in large number in sections from 25 canine carcinomas. These cells formed small clusters at the margins of the tumour. There were significantly more IgG+ cells and IgA+ cells in transitional carcinomas than in adenocarcinomas. The number of IgA+ cells in canine nasal carcinomas was significantly greater than the number of IgG+ cells.
Taken together these findings suggest that the dog makes an ineffective T-cell mediated immune response to nasal carcinoma which may in part account for the malignant potential of these lesions. The strongest cellular immune response, involving T cells, MHC class II+ APC and IgG plasma cells is associated with transitional carcinoma. Functional (cytokine and chemokine gene expression) studies have not yet been completed on this series of lesions.
The work described in this presentation is the result of a long-term collaboration between the author and colleagues at the University of Liège in Belgium, in particular Professor Cecile Clercx, Dr Dominique Peeters, Dr Fred Billen and Ms Morgane Vanherberghen.
A list of further reading can be supplied on request.