W. Baumgärtner

Immunohistochemical Characterization of Inflammatory Cells in Brains of Dogs with Granulomatous Meningoencephalitis

The inflammatory cells of eleven dogs with canine granulomatous meningoencephalitis were characterized immunohistochemically. Macrophages were identified by antibodies directed against lysozyme and the DH82 antigen (expressed by cells of a malignant histiocytosis). T cells were demonstrated by CD3, CD43, and CD45R antigen, and B cells by immunoglobulin G and immunoglobulin M expression. Furthermore, staining for the major histocompatibility complex (MHC) class II antigen was evaluated. Diseased animals ranged from 1 to 9 years of age. Small and medium-sized breeds were affected predominantly. Lesions were widespread and localized mainly in the brain stem, less frequently in the cerebrum or cerebellum. Alterations were represented by perivascular cuffs, parenchymal granulomas, and leptomeningeal infiltrates. Lymphocytes and macrophages comprised the dominant cell populations; their percentage varied substantially between different animals and between sections from the same individual. Immunohistochemically, the bulk of lymphocytes were CD3 antigen-positive T cells, while only a few cells were CD43 and CD45R antigen-positive or were classified as B cells. The majority of macrophages expressed both lysozyme and DH82 antigen; however, some were positive for only one antigen. MHC class II antigen-expression, observed only within and in close proximity to the lesions, was found on all inflammatory cells, pericytes/endothelial cells, and microglia. Results were negative for canine distemper virus antigen and nucleoprotein mRNA, rabies virus antigen, fungi, bacteria, and protozoal agents. This immunomorphologic study reveals that inflammatory lesions in canine granulomatous meningoencephalitis consist of a heterogeneous population of MHC class II antigen-positive macrophages and predominantly CD3 antigen-positive lymphocytes. The data suggest a T cell-mediated delayed-type hypersensitivity of an organ-specific autoimmune disease as a possible pathogenic mechanism for this unique canine brain lesion.

Identification of CD4+ and CD8+ T cell subsets and B cells in the brain of dogs with spontaneous acute, subacute-, and chronic-demyelinating distemper encephalitis

CD4 and CD8 antigen expression of T cells as well as B cell and canine distemper virus (CDV) antigen distribution were immunohistologically examined in the cerebellum of dogs with spontaneous distemper encephalitis. Cellular and viral antigen expression were evaluated at intralesional and extralesional sites and in the perivascular space. Histologically, acute and subacute non-inflammatory encephalitis and subacute inflammatory and chronic plaques were distinguished. Demyelination was a feature of all subacute and chronic lesions, although the majority of plaques exhibited no or only a low level of active demyelination as demonstrated by single macrophages with luxol fast blue positive material in their cytoplasm. CDV antigen expression, observed in all distemper brains, was reduced in chronic plaques. CD4+, CD8+, and B cells were absent in controls and in some brains with acute encephalitis. A mild infiltration of CD8+ cells was noticed in the neuropil of the remaining brains with acute and all brains with subacute non-inflammatory encephalitis. Single CD4+ cells were found in two brains with acute and in all brains with subacute non-inflammatory encephalitis. Numerous CD8+ and CD4+ cells and few B cells, with a preponderance of CD8+ cells, were detected in subacute inflammatory and chronic lesions. In contrast, in perivascular infiltrates (PVI) of subacute and chronic lesions a dominance of CD4+ cells was detected. The dominating CD8+ cells in acute and subacute non-inflammatory encephalitis might be involved in viral clearance or contribute as antibody-independent cytotoxic T cells to early lesion development. In subacute inflammatory and chronic lesions CD8+ cells may function as cytotoxic effector cells and CD4+ cells by initiating a delayed-type hypersensitivity reaction. The simultaneous occurrence of perivascular B and CD4+ cells indicated that an antibody-mediated cytotoxicity could synergistically enhance demyelination. Summarized, temporal and spatial distribution of CD4+, CD8+ and B cells and virus antigen in early and late lesions support the hypothesis of a heterogeneous in part immune-mediated plaque pathogenesis in distemper demyelination.

Increased expression of pro-inflammatory cytokines and lack of up-regulation of anti-inflammatory cytokines in early distemper CNS lesions.

To investigate the pathogenesis of early lesions in canine distemper virus (CDV) leukoencephalomyelitis, the expressions of pro- and anti-inflammatory cytokines such as interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and transforming growth factor (TGF)-beta and the housekeeping genes beta-actin and GAPDH were studied using semi-quantitative RT-PCR. Relative cytokine values were related to the degree of CDV infection, MHC class II expression and infiltration of CD4-, CD8- and CD3epsilon-positive lymphocytes. Actin up-regulation, in contrast to GAPDH, was influenced by CDV infection and therefore could not be used as an internal standard to study cytokine expression. In early CDV infection of the cerebellum, either no detectable lesions or mild infiltration of CD8 positive cells or demyelination and up-regulation of MHC class II antigen were observed. IL-6, -8, -12 and TNF-alpha transcripts were found in 94%, 94%, 78% and 56% of distemper dogs, respectively, compared to 17%, 33%, 0% and 0% in controls, whereas IL-1beta, -2 and IFN-gamma were not detectable in any of the studied cerebella. Conversely, IL-10 and TGF-beta transcripts were present in 83% and 100% of the investigated cerebella of distemper dogs and controls. Relative RT-PCR results, expressed as %GAPDH, revealed a significant up-regulation of IL-6, -8, -12 and TNF-alpha mRNA in distemper dogs; whereas IL-10 and TGF-beta showed only a weak and not significantly increased expression following infection. Relative pro-inflammatory cytokine expression values were highest following CDV infection, indicating that the virus itself directly triggered the up-regulation of the pro-inflammatory cytokines. Succeeding changes, such as lymphocyte infiltration, MHC class II up-regulation and demyelination resulted only in a minor additional increase in cytokine expression, implying a secondary or by-stander mechanism of cytokine activation by these changes. Disease initiation and progression in early distemper leukoencephalomyelitis seemed to be due to a lacking or inappropriate response of the anti-inflammatory cytokines in the presence of a vigorous up-regulation of pro-inflammatory cytokines.

Phenotypical characterization of T and B cell areas in lymphoid tissues of dogs with spontaneous distemper.

CD3, CD4, CD5, and CD8 antigen expression of T cells and IgG expression of B cells and canine distemper virus (CDV) antigen distribution were immunohistochemically examined in lymphoid tissues (lymph node, spleen, thymus, and tonsil) of control dogs and animals with spontaneous canine distemper. In addition, CNS tissue of all animals was studied for neuropathological changes and CDV antigen distribution. Based on the degree of depletion distemper dogs were classified into two groups. Group I represented animals with moderate to marked lymphoid depletion, while group II dogs displayed mild or no depletion. CDV antigen was mainly found in lymphocytes and macrophages of group I dogs, whereas CDV expression was most prominent in dendritic cells of group II animals. In group I dogs, a marked loss of CD3, CD4, CD5, CD8, and IgG expression was noticed, hereby loss of CD4+ cells was more prominent than depletion of CD8+ cells. In the lymphoid tissues of group II animals, a significant increase in the number of T and B cells was observed compared to group I dogs. The number of CD3+, CD4+, and CD8+ cells in group II dogs was similar to the findings in controls, however, CD5 and IgG expression was mildly reduced in T and B cell areas, respectively. Additionally, in groups I and II dogs, CD3+ and CD5- T cells were detected in T cell areas. Whether this cell population represents a cell type with autoimmune reactive potential remains to be determined. Surprisingly in group II animals, viral antigen was found predominantly in dendritic cells indicating a change in the cell tropism of CDV during chronic infection and a possible mechanism of viral persistence. The two patterns of lymphoid depletions correlated to two different types of canine distemper encephalitis (CDE). Group I dogs displayed acute non-inflammatory CDE, whereas group II dogs suffered from chronic inflammatory demyelinating CDE, indicating a pathogenic relationship between lymphocytic depletion and inflammatory brain lesions in distemper.

Cytokine mRNA expression in whole blood samples from dogs with natural canine distemper virus infection

Cytokines are soluble polypeptides with many physiological functions and a special role during infection and inflammation. Little is known about cytokine regulation in naturally occurring viral diseases of animals. Especially the role of cytokines in the development and progression of lesions in canine distemper virus (CDV) infection in dogs is largely unknown. Whole blood samples from 14 dogs with CDV infection and three dogs suffering from non-distemper diseases were examined for mRNA of pro-inflammatory cytokines such as interleukin-1beta (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and the anti-inflammatory transforming growth factor-beta1 (TGF) using reverse transcription polymerase chain reaction (RT-PCR). Blood samples from the three dogs that showed no clinical abnormalities during a pre-vaccination physical examination served as control. CDV infection was confirmed by post-mortem immunohistochemistry for CDV nucleoprotein. The degree of immunoreactivity and the number of virus antigen positive organs were expressed as antigen index. IFN transcripts were not identified in any dog and IL-8 transcripts were present in RNA isolates from all 20 dogs. None of the other cytokines was detected in control animals. IL-1 and IL-6 were each found in one non-distemper dog and TGF transcripts were amplified in two dogs with non-distemper disease. The following transcripts were found in variable numbers in distemper dogs: IL-1 (7/14 dogs), IL-6 (3/14 dogs), IL-12 (3/14 dogs), TNF (8/14 dogs), and TGF (10/14 dogs) with multiple cytokines in ten dogs. No cytokine transcripts were detected in three distemper dogs. There was no obvious correlation between cytokine mRNA expression and respiratory and gastrointestinal tract diseases. In the CNS, demyelination was frequently associated with IL-1, IL-12, TNF and TGF mRNA expression in the blood. IL-6 transcripts were found only in animals with early CNS lesions and TGF was the only detectable cytokine in an animal with chronic demyelination. Lack of detectable cytokine transcripts in whole blood samples was associated with a high antigen index and viremia, indicating that an overwhelming virus infection may suppress cytokine production, possibly due to paralysis of the immune system. Simultaneous occurrence of pro- and anti-inflammatory cytokines in whole blood preparation from most of the dogs with distemper, indicated a complex most likely disease stage dependent orchestrated cytokine expression.

Dominating interleukin-10 mRNA expression induction in cerebrospinal fluid cells of dogs with natural canine distemper virus induced demyelinating and non-demyelinating CNS lesions.

Canine distemper virus (CDV) infection in dogs is commonly associated with demyelinating leukoencephalitis (DL). Although the mechanism of primary demyelination in distemper remains undetermined recent studies showed a direct virus-induced cytolysis in early non-inflammatory and immune-mediated mechanisms in inflammatory lesions. To further investigate the pathogenesis of this morbillivirus-induced demyelination the expression of a variety of cytokine mRNA species (interleukin (IL)-1beta, IL-2, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and interferon (IFN)-gamma in cerebrospinal fluid cells of 12 dogs with CDV encephalitis was investigated employing reverse transcription-polymerase chain reaction (RT-PCR) and these findings were correlated to the type of CNS lesions. Neuropathology revealed the whole spectrum of distemper DL lesions from acute to chronic alterations, however, most plaques lacked active demyelination. Three control animals were devoid of any cytokine expression, whereas in distemper animals IL-10 transcripts were found in nine dogs with acute and chronic lesions. IL-6, TNF, and TGF mRNA was found in six, four, and three animals, respectively. IL-12 and IFN-gamma, suggestive of a TH1-like dominated immune response, were detected only in one animal with chronic lesions. Summarized, TNF and IL-6, associated with disease exacerbation, and IL-10 and TGF, indicative of remission, were often observed simultaneously in distemper DL and could not be assigned to a specific disease stage. However IL-10 mRNA remained the most frequently detected cytokine indicating a stage of inactivity in most animals investigated.

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques.

Abstract CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-μm-thick serial sections were stained with Hu2002&u2002E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection.

Interleukin-1β, -6, -12 and tumor necrosis factor-α expression in brains of dogs with canine distemper virus infection

Abstract Canine distemper virus infection in dogs is commonly associated with demyelinating central nervous system lesions. Investigations on viral protein expression by studying mRNA and protein distribution together with the characterization of CD4 and CD8 inflammatory cells and MHC class II up-regulation revealed a biphasic disease process. To further investigate the cellular interactions in the different plaque types the cerebella of 14 dogs with confirmed distemper infection were investigated for expression of interleukin (IL)-1β, -6, -12 and tumor necrosis factor-α (TNF) by immunohistochemistry using rabbit polyclonal anti-cytokine antibodies. T-cells and astrocytes were identified with rabbit anti CD3- and GFAP-monoclonal and polyclonal antibodies, respectively; and microglia/macrophages were characterized by their ability to bind lectin from Bandeiraea simplicifolia (BS-1). To further name the cytokine expressing cells immunoenzymatic double staining using DAB and New Fuchsin was performed. White matter lesions were classified according to histopathological criteria into acute, subacute and chronic. Canine distemper virus nucleoprotein antigen was demonstrated in nearly all plaques, except in older plaques where virus was not present within the plaque but adjacent to the lesion. IL-1 expression was observed to varying degrees in all types of lesions. Most often IL-1 was present in CD3 and BS-1 positive cells in the brain parenchyma in earlier plaques and comprising perivascular cuffs found in chronic plaques. IL-6 expression was present in all lesions, and followed a similar distribution pattern as IL-1. IL-12 displayed very often a granular extracellular pattern of immunoreactivity, especially in the brain parenchyma, and was found only in individual perivascular cells. TNF staining, predominantly found in astrocytes, was present in lesions of various types; however, staining appeared to be stronger in acute lesions and decreased in chronic plaques. In the latter, TNF seemed to be more prominent in areas adjacent to the plaques. Summarizing, in early non-demyelinating lesions without overt inflammation TNF seemed to be important, whereas in distemper lesions with inflammatory infiltrates IL-1 and to a lesser degree IL-6 were more prominent. These results imply that TNF may be involved in the pathogenesis of early demyelination in nervous distemper.

Immunopathological studies on feline cutaneous and (muco)cutaneous mycobacteriosis

Eight cases of feline (muco)cutaneous mycobacteriosis were studied to identify the causative agent and examine for phenotype and functional characteristics (expression of interleukin (IL)-1beta, IL-6, IL-12, tumour necrosis factor-alpha and inducible nitric oxide synthase) of the inflammatory cells. Polymerase chain reaction and sequencing identified the causative agents as Mycobacterium tuberculosis or M. avium complex in each four cases. Lesions were characterised by pyogranulomatous infiltration, with variability in the presence and size of necrotic areas, the presence of multinucleated giant cells and the degree of lymphocyte infiltration. Macrophages were positive for myeloid/histiocyte antigen (calprotectin), suggesting they represented freshly recruited monocytes; further differentiation to epithelioid cells and multinucleated giant cells was associated with loss of the myeloid/histiocyte antigen. Lymphocytes were found disseminated in the infiltrate (predominantly T cells) and as B cell-dominated accumulations mainly in the periphery of the lesions. Acid-fast bacilli were numerous. In M. tuberculosis complex infection, extracellular bacilli were most prominent, whereas in M. avium complex infection, bacilli were mainly located intracellularly. All cytokines examined as well as inducible nitric oxide synthase (iNOS) were variably expressed by macrophages, epithelioid cells and multinucleated giant cells. Expression was most intense in degenerating macrophages loaded with intracellular bacilli, but was also seen cell-free within necrotic areas. The intense induction of cytokine and iNOS expression especially in infected macrophages suggests a relatively low virulence for these infectious agents in cats. Furthermore, the confinement of the bacilli to lesions indicates a successful response to infection.

EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II ANTIGEN IN NEOPLASTIC CELLS OF CANINE CUTANEOUS HISTIOCYTOMA

Forty five cases of canine cutaneous histiocytoma (CCH) were examined by immunohistology for expression and distribution of major histocompatibility complex (MHC) class II antigen in neoplastic cells. In addition, expression of lysozyme and calprotectin (leucocyte protein L1) in neoplastic cells was investigated. Furthermore, B and T lymphocytes were demonstrated by antibodies against the CD3 antigen, IgG, and IgM. Neoplastic cells showed two staining patterns for MHC class II antigen: focal juxtanuclear cytoplasmic staining and/or rim-like staining along the cell periphery. In 24 cases, a predominant or exclusive focal juxtanuclear cytoplasmic MHC class II antigen reaction in neoplastic cells, and the presence of few diffusely distributed infiltrating CD3 antigen-positive T lymphocytes were observed. Tumors with numerous neoplastic cells exhibiting staining for MHC class II antigen along the cell periphery (n = 21) showed increased inflammatory alterations, represented by disseminated and nodular infiltrations of mainly CD3 antigen-positive T cells. B cells, plasma cells, exudate macrophages, and neutrophils were rarely seen disseminated between neoplastic cells whereas their number increased within focal inflammatory infiltrates. The focal cytoplasmic reaction for MHC class II antigen in neoplastic cells might represent newly synthesized MHC class II molecules stored in vesicles, whereas staining of the cell periphery might occur due to accumulation of MHC class II molecules along the plasma membrane. The increasing expression of MHC class II molecules on the cell surface might be the decisive factor for onset and progression of tumor regression. However, the exact mechanism of priming and activation of T cells by neoplastic cells and the nature of the presented antigen are not yet known.