The influence of breed, age, gender and methylprednisolone on the distribution of lymphocyte subpopulations in dog

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Aus der Klinik für Kleine Haustiere der Tierärztlichen Hochschule Hannover

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The influence of breed, age, gender and methylprednisolone on the distribution of lymphocyte subpopulations in the dog: a flow cytometric study

INAUGURAL – DISSERTATION Zur Erlangung des Grades eines Doktors

der Veterinämedizin (Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von Jetsada Rungpupradit

aus Bangkok Thailand

Hannover 2003

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Wissenschaftliche Betreuung: Univ.-Prof. Dr. I. Nolte

1. Gutachter: Univ.-Prof. Dr. I. Nolte 2. Gutachter: Univ.-Prof. Dr. M. Kietzmann

Tag der mündlichen Prüfung: 23. September 2003

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my parents

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Contents

1 Introduction 9

2 Literature review 11

2.1 Lymphocytes 11

2.1.1 T lymphocytes 11

2.1.1.1 T helper cells 12

2.1.1.2 T cytotoxic cells 12

2.1.2 Natural killer cells 13

2.1.3 B cells 13

2.2 Monoclonal antibodies 14

2.3 Breed susceptibility in immunodeficiency disease and cancer incidence 14 2.4 Lymphocyte subsets in peripheral blood of healthy dogs 16 2.5 The influence of breed on peripheral blood lymphocytes in healthy dogs

16 2.6 The influence of age on peripheral blood lymphocytes in healthy dogs 17 2.7 The influence of gender on peripheral blood lymphocytes in healthy dogs

19 2.8 Effect of corticosteroids on peripheral blood lymphocyte subsets 20

3 Materials and Methods 23

3.1 Materials 23

3.1.1 Animals 23

3.1.2 Antibodies 25

3.1.3 Reagents 27

3.2 Methods 28

3.2.1 Sample collection 28

3.2.2 Effect of methylprednisolone on peripheral blood lymphocyte subsets 28

3.2.2.1 Experiment 1 28

3.2.2.2 Experiment 2 29

3.2.3 Staining of cells 30

3.2.4 Flow cytometry measurements 31

3.2.5 Data analysis 31

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3.2.6 Immunophenotyping data report 34

3.2.7 Statistical Analysis 34

4 Results 35

4.1 Reproducibility test 35

4.2 Gate purity and recovery 36

4.3 Lymphocyte subpopulations in peripheral blood of healthy dogs 36 4.4 Influence of breed on lymphocyte subset distribution 37 4.5 Influence of age on lymphocyte distribution 48 4.6 Influence of gender on lymphocyte distribution 62 4.7 Effect of methylprednisolone on peripheral blood lymphocyte subsets 65

4.7.1 Experiment 1 67

4.7.2 Experiment 2 68

5 Discussion 99

6 Summary 109

7 Zusammenfassung 111

8 References 113

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Abbreviations

ALT alanine aminotransferase

AP alkaline phosphatase

APC antigen presenting cell

BE Beagle

BMD Bernese mountain dog

ca canine

CBC complete blood count

CD cluster of differentiation

C.V. Coefficient of Variation

D day

f female

Fab fragment antigen binding

FC flow cytometry

FCR Flat-coated Retriever

FITC Fluoresceinisothiocyanat

fs spayed female

FSC forward scatter

GLDH glutamate-dehydrogenase

Ig immunoglobulin

IL interleukin

M month

m male

mAb monoclonal antibodies

MBD mixed breed dog

MHC major histocompatibility complex

min minute

mk castrated male

PB peripheral blood

PE Phycoerythrin

PerCP Peridin-Chlorophyll-a-Proteincomplex

Q Quartile

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R region

RT room temperature

RW Rottweiler

SmIg surface membrane immunoglobulin

SSC side scatter

STD Standard Deviation

TCR T cell receptor

TGF Transforming growth factor

WM Weimaraner

Y year

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Introduction

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1 Introduction

In recent years, clinical application of flow cytometry has become increasingly important in the evaluation of immunopathies and hematologic malignancies in companion animal medicine (TOMPKINS et al., 1991; CHABANNE et al., 2000; CULMSEE, 2000; CULMSEE et al., 2001; CULMSEE and NOLTE, 2002). The utility of lymphocyte subset quantitation by flow cytometry for the classification of diseases however depends on the determination of reference values in healthy individuals. In human medicine a substantial number of studies have focused on healthy subjects to establish normative data, and matched reference ranges have been published taking into account the recognized influence of physiological parameters such as age, gender, and race or ethnical background on the absolute and relative numbers of circulating lymphocyte subsets (ERKELLER-YUKSEL et al., 1991; HUSTAERT et al., 1994;

SHAHABUDDIN et al., 1998). In veterinary medicine only some researchers have reported reference values for peripheral blood lymphocyte subpopulations in dogs of different breeds (BYRNE et al., 2000; CULMSEE 2000; FALDYNA et al., 2001). However, the total number of healthy dogs included in those studies was rather low. Another drawback to the use of the reference values of the studies cited is that methods, antibodies, sample processing and animals differed and are thus difficult to compare. Therefore one main intention of this study was the definition of reference ranges of relative and absolute numbers of lymphocyte subsets by evaluating a large cohort of healthy dogs. Moreover, the influence of breed, age and gender using a standard protocol to reduce variability in both sample preparation technique and flow cytometer methodology was investigated.

There are some canine breeds that are known to have a predilection for specific diseases of the immune system or to be specially prone to develop tumor diseases (DAY, 1999;

COOLEY et al., 2002; MORRIS et al., 2002). The pathogenesis of these diseases is more or less unclear, however it must be assumed that either the cellular or the humoral specific immunity is somehow altered. As there is growing evidence that, like in man and other species, the distribution of lymphocyte subpopulations of different canine breeds varies also in healthy individuals (FRANZ, 1996; BYRNE et al., 2000; FALDYNA et al., 2001), for the diagnosis, and especially for understanding the pathogenesis of the diseases, the standard for

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Introduction

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these breeds needs to be determined. Therefore, it was an aim of this study to define representative breed specific reference values for some selected breeds known to suffer particularly often from immunological diseases and tumors. The breeds chosen were the Rottweiler, Weimaraner, Flat-coated Retriever and Bernese mountain dog. Also, a large group of mixed breed dogs and Beagles were investigated for determination of a standard reference range and to allow for comparison with the values reported in the literature.

Immunsuppressive drugs are widely used in the therapy of immune diseases and also of tumors, especially hematological malignancies in the dog. Despite their frequent use, astonishingly little is known about the influence these drugs have on lymphocyte subpopulations (COHN, 1997; MILLER, 1997; DAY, 2002). A precise characterization of this influence would, however, be especially useful in the assesment of pretreated referred patients, where it is often unclear if the immune status is altered due to the underlying disease or due to a preceding therapy. The second part of the study therefore consisted of the flow cytometrical investigation of lymphocyte subsets after the administration of methylprednisolone in healthy Beagles.

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Literature review

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2 Literature review 2.1 Lymphocytes

Lymphocytes consist of heterogeneous populations of cells that differ greatly from each other in terms of origin, life span, preferred areas of settlement within organs, surface structure and function. Although some morphologic characteristics such as size, granularity, and nucleocytoplasmatic ratio distinguish lymphocyte populations from each other, they provide no clues of either lineage or function (FERGUSON, 1994). The most important, precise, and quantitative method now in use in veterinary clinical laboratories is based on the identification of certain glycoproteins displayed on the membrane of lymphocytes and collectively denominated markers or clusters of differentiation (COBBOLD and METCALFE, 1994; CULMSEE, 2000). The identification of patterns in these cell surface molecules has allowed the identification of two large immunphenotypically and functionally different lymphocyte classes. T lymphocytes, which develop in the thymus, are responsible for cell-mediated immunity and B lymphocytes, which in mammals develop in the adult bone marrow or the fetal liver, produce antibodies and are therefore considered to be the carriers of humoral immunity. (PAUL, 1984; KUBY, 1992).

2.1.1 T lymphocytes

T lymphocytes (T cells) originate from lymphoid stem cells in the bone marrow that migrate to mature in the thymus gland (STARR et al., 2002). T cells are defined by the expression of a T cell receptor (TCR) complex. The TCR complex consists of two different glycosylated polypeptide chains and a series of minor components (CD3), that act as signal transducers (FEITO et al., 2002; KELLERMANN et al., 2002). In dogs the majority of T cells have an α and a β chain forming the TCR complex, whereas a minority of T cells have γδ TCRs (TIZARD, 2000). In contrast, about 60 % of ruminant T cells have been shown to have γδ TCRs, suggesting that species specific differences exist (TIZARD, 2000).

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The diverse specificity of TCRs is achieved by variable genetic combination of an array of gene segments (denominated V, D and J) that encode portions of the TCR chains (KUBY, 1992). T cells with αβ TCRs can be further subdivided into functionally different T helper/inducer cell and T cytotoxic/suppressor cell subpopulations based on membrane expression of CD4 and CD8 markers.

2.1.1.1 T helper/inducer cells

T helper cells express CD4 molecules and recognize antigenic peptides in association with class II MHC molecules (FORMAN, 1984). These CD4+ T cells are activated by interaction with an antigen -MHC (major histocompatibility complex) class II complex on the surface of antigen-presenting cells (APC) and various cytokines including interleukin 1 (IL-1) released by the latter cells. IL-1 binds to a specific receptor on CD4+ T cells and thereby induces them to secrete IL-2 as well as to express IL-2 receptors (WEAVER and UNAUE, 1990). The CD4+ T cells thus stimulated divide (clonal proliferation) and further differentiate (MARY et al., 1987). Activated CD4+ T cells secrete a variety of other cytokines, called lymphokines, that play an important role in the activation of B cells, CD8+ T cells, and numerous other cells that are involved in the immune response (BALKWILL and BURKE, 1989). Depending on the kind of cytokines produced, CD4+ T cells can be subdivided into Th-1 and Th-2 subsets (MOSMANN and COFFMANN, 1989; ABBAS et al., 1996). Th-1 cells produce IL- 2, interferon-gamma (IFN-γ), and tumor necrosis factor beta (TNF-β), which promote cellular immune response. Th-2 cells produce IL-4, IL-5, IL-6, IL-10 and transforming growth factor (TGF), which promotes antibody synthesis by B cells (HODGKIN et al., 1991).

2.1.1.2 T cytotoxic/suppressor cells

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surface of altered self-cells (TAKAI et al., 1986; MARASKOVSKY et al., 1989). CD8+ T cells play an important role in the killing of virus infected cells and tumor cells, as well as in host-versus-graft immune reactions (GUIDOTLI et al., 1996; OLIVER et al., 1999;

KRUPNICK et al., 2002).

2.1.2 Natural killer cells

At light microscopy natural killer (NK) cells are characterized by their large size and distinct cytoplasmatic granules. Their membrane lacks the cell surface markers of T cell (TCR) or B cell (SmIg) lineage (TIZARD, 2000). In humans, immunophenotypical surface markers used to identify NK cells include CD16 and CD56 (BARCLAY et al, 1997). Their functional importance resides in tumor surveillance and killing of virus infected cells (PAUL, 1984). Alternatively, they can induce the lysis of cells in the presence (antibody-dependent cell mediated cytotoxicity [ADCC]) or absence of antibodies (BONNEMA et al., 1994). In addition to their killing property, NK cells secrete several cytokines, such as IFN-γ, that promotes Th-1 and inhibits Th-2 immune responses (PERNIS et al., 1995).

2.1.3 B lymphocytes

B lymphocytes (B cells) originate from hematopoietic stem cells, and mature and differentiate in the bone marrow (HENDERSON and DORSHIKIND, 1990). B cells are responsible for the humoral response of the immune system. To produce antibodies, B cells proliferate and differentiate to plasma cells after interacting with both antigen and CD4+ T cells (FINKELMANN et al., 1992; KUBY 1992; PARKER 1993). B cells are defined by the expression of surface membrane immunoglobulins (SmIg), and cell surface markers commonly used in human medicine to identify them include include CD19, CD20, CD21 and CD22 (BARCLAY et al., 1997; SHAW et al., 1998). In dogs, use of CD21 has gained widest acceptance among researchers.

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2.2 Monoclonal antibodies

In the past, the limited number of available species specific monoclonal antibodies significantly restricted the use of lymphocyte immunophenotyping in veterinary medicine. In 1993, the First International Canine Leukocyte Antigen Workshop, CLAW (COBBOLD and METCALFE, 1994), was conducted to identify canine leucocyte antigens, and monoclonal antibodies that recognize homologous antigens were classified in analogy to the human and murine cluster of differentiation (CD) nomenclature. Since then, cross reactivity of several anti-human lymphocyte antibodies to lymphocyte surface antigens in domestic animals has been proved in several publications (GREENLEE et al., 1987; CHABANNE et al., 1994;

SCHUBERTH et al., 1998; CULMSEE, 2000). These studies, together with the increasing commercial availability of species specific monoclonal antibodies for companion animals have contributed to establish immunophenotyping as an attractive and important tool for researchers in the field of clinical immunology (TOMPKINS et al., 1990; GEBHARD and CARTER, 1992; MOORE et al., 1992; VOSS et al., 1993).

2.3 Breed susceptibility in immunodeficiency diseases and cancer incidence

Primary immune mediated diseases are uncommonly reported in dogs, and specific immunological defects in such conditions are, to date, poorly characterized. These disorders are usually inherited, and breed related susceptibility is believed to be related to selective breeding for desired traits. Affected animals suffer from chronic recurrent disease involving the respiratory and alimentary tracts, joints, skin, peripheral lymph nodes, central nervous system and conjunctivae (DAY, 1997). Among the breeds for which primary immune mediated diseases have been reported are German Shepherd dogs (DAY and PENHALE, 1988; BATT et al., 1991; CHABANNE et al., 1995), Grey Collies (DALE et al., 1995), Irish

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genetically predisposed to immunodeficiency diseases, although the precise nature of the defect or defects remains to be elucidated. Low serum immunoglobulin concentrations in related Weimaraners have been reported (DAY et al., 1997), and evidence of abnormal in vitro neutrophil metabolism in response to stimulation (COUTO et al., 1989) and neutrophil phagocytic dysfunction (HANSEN et al., 1995) have been documented. Likewise, many cancers have been found to have breed predilection. Osteosarcoma is diagnosed more often in giant breeds including Rottweilers (COOLEY et al., 2002), and soft tissue sarcomas and malignant histiocytosis have a high incidence among Flat-coated Retrievers (MORRIS et al., 2000; MORRIS et al., 2002) and Bernese mountain dogs (MOORE, 1984; ROSIN et al., 1986; PADGET et al., 1995; PATERSON et al., 1995).

At present, the establishment of cancer in a host is thought to involve at least two major events. First, malignant tumors are believed to arise as a result of a series of specific genetic events which include the activation of oncogenes and/or the loss of function of tumor suppressor genes. A large array of control mechanisms is operative within a cell to prevent abnormal growth, and if these fail cells continue to proliferate. Subsequent interaction of the malignant cell with its microenvironment, including the immune system, may result in tumor progression or regression. Second, the escape of tumor cells from immunological recognition seems to play a central role in the development of cancer (YOSHIZAWA et al., 2001). The underlying premise of tumor immunology is that the immune system is capable of recognizing cancer cells and that immune recognition can lead to the elimination of tumors by the host (BURNET, 1970). The growth and metastatic spread of tumors, to a large extent, depend on their capacity to evade host immune surveillance and overcome host defenses mechanisms (FOSS, 2002). Despite an increasing understanding of immunological mechanisms it is still not clear how tumors evade immune-surveillance of the host and how tumors interact with the immune system. In particular, the question whether tumors arise because of immunodeficiency or whether tumor cells employ active strategies to escape the control of the immune system is still not answer (SALIH and NUSSLER, 2001). Both the lack of specific tumor antigen and down-regulation of major histocompatibility complex (MHC) molecule expression hamper recognition of neoplastic cells by T lymphocytes (GILBOA, 1999; OCHSENBEIN, 2002). In the presence of defective expression of ligands

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for the T cell costimulatory receptors, tumor recognition may lead to the development of tolerance instead of specific cytotoxic activity (BANAT et al., 2001). Tumor cell counter- attack against effector T cells has also been described, using either inhibitory cytokines (ANTONIA et al., 1998; , MOCELLIN et al., 2001), induction of apoptosis via Fas signalling (WALKER et al., 1997), functional inactivation through disruption of normal CD40/CD40 ligand interactions (TONG et al., 2001), or induction of anergy (WHITESIDE, 2003).

2.4 Lymphocyte subsets in peripheral blood of healthy dogs

Reference ranges are necessary in clinical chemistry and hematology for correct assessment of measurements. Likewise, reference ranges of the relative and absolute numbers of peripheral blood lymphocyte subsets need to be defined in order to evaluate the immune status, and distinguish normal variability from disease. In human medicine numerous studies have been focused on lymphocyte subsets in healthy individuals to establish reliable reference ranges that account for the normal physiological variability observed between individuals of different age, gender, and race or ethnical origin (ROMAN et al., 1995; MCCLOSKEY et al., 1997; SANTAGOSTINO et al., 1999).

In veterinary medicine, reference ranges for peripheral blood lymphocyte subsets in healthy dogs have been published for a limited number of breeds by FRANZ (1996), GREELEY et al. (1996), BYRNE et al. (2000), CULMSEE (2000), FALDYNA et al. (2001), and TOMAN et al. (2002). Comparable studies have also been performed in other domestic animals including cats (TOMPKINS et al., 1991; ENGLISH et al., 1994; SELLON et al., 1996) and horses (MCFARLANE et al., 2001).

2.5 Influence of breed on the peripheral blood lymphocytes in healthy dogs

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(HOWARD et al., 1992). Since the public health importance of the AIDS pandemics accentuated the need for reliable reference ranges, considerable effort has been made to study the influence of race or ethnical background on circulating lymphocyte subsets in healthy individuals (SENJU et al., 1991; UPPAL et al., 2003). In healthy humans, lower percentages of T cells and CD4+ T cells, and higher percentages of NK cells have been reported in Asians (CHOONG et al., 1995; HOWARD et al., 1996; KAM et al., 1996, VITHAYASAI et al., 1997). African-Americans had significantly lower absolute counts of CD8+ T cells than Hispanics and non-Hispanics (RUDY et al., 2002). A low percentage of CD4+ T cells and high percentages of CD8+ T cells has been reported in the Ethiopian population (TSEGAYE et al., 1999). Blacks had a higher proportion of B cells and lower proportion of T cells than whites according to TOLLERUD (1989).

Analogous investigations in healthy dogs to determine the influence of breed specific genetic factors on peripheral blood lymphocyte subpopulations have been done. One study reported Terriers and Labrador retrievers to have lower percentages of CD8+ T cells than Beagles and mixed breed dogs (BYRNE et al., 2000). FALDYNA et al. (2001), investigating lymphocytes and their subsets in adult Beagles, German Shepherds, Dalmatians, and Dachshunds documented higher percentages in Beagles and Dachshunds than in Dalmatians and German Shepherds. Highest and lowest absolute lymphocyte counts were found in Beagles and German Shepherds, respectively. Consequently, German Shepherds showed the lowest absolute counts of CD3+, CD4+, CD8+ and CD21+ cells. Dalmatians had the lowest percentage numbers of CD3+ cells, the highest percentage of CD21+ cells, and the lowest CD4/CD8 ratio. German Shepherds showed the lowest percentage of CD21+ cells and the highest CD4/CD8 ratio. In agreement with Faldyna`s findings in Dalmatians, the study from FRANZ (1996) also found a low percentage of CD5+ T cells in this breed when compared to German Shepherds, Terriers, Poodles and mixed breed dogs.

2.6 Influence of age on the peripheral blood lymphocytes in healthy dogs

Advancing age is accompanied by a variety of alterations in the immune system, and the existence of a relationship between advancing age and a decline in the total leucocyte and

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absolute lymphocyte counts has been known to scientists for more than 60 years (KATO, 1935). The term immunosenescence has been coined to describe the sum of dysregulations of the immune system and its interaction with other systems which is believed to manifest itself in the increased susceptibility to cancer, autoimmune diseases and infectious diseases of old age (BANKS 1981; MORRISON and OTT 1981; HIROKAWA, 1992; HORIUCHI and WILMOTH, 1997; PAWELEC et al., 1997). Thymic involution, decreased T cell response to antigenic and non-specific activation stimuli, decreased activation-induced intracellular phosphorylation, altered cytokine expression by T cells, and accumulation of senescent memory T cells all have been considered to contribute to age–related immune defects (ROBERTS-THOMSON et al., 1974; MURASKO et al., 1988; HORIUCHI and WILMONT- H 1997; PIDO-LOPEZ et al., 2001;). In addition to these intrinsic factors, factors outside the immune system are known to play an important role in the continuous restructuring process of the immune system (GLOBERSON and EFFROS, 2000). Among these extrinsic factors, hormones are the most important and exert their influence on immune response and cytokine environment either directly through surface receptors located on lymphocytes and antigen presenting cells, or indirectly through their recognized influence on thymic mass and function (ASPINALL 1997; LACORAZZA et al., 1999).

In humans, a decrease in the absolute counts of leucocytes, lymphocytes, T, B, NK, CD4+

and CD8+ cells have been found associated with increasing age by most authors (ERKELLER-YUKSEL et al., 1991; KOTYLO et al., 1992; SANSONI et al., 1993;

HULSTAERT et al., 1994), although WIENER et al. (1990) found no significant correlation between age and absolute numbers of CD4+ and CD8+ lymphocytes. Relative counts of B cells decline (HULSTAERT et al., 1994) whereas NK cells either decline (ERKELLER- YUKSEL et al., 1991) or remain unchanged (SHAHABUDDIN et al., 1998). Percentage numbers of T, CD4+ and CD8+ cells were found to increase by some investigators (WIENER

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Similar to humans, in dogs a decrease in absolute counts of lymphocytes, T, B, CD4+ and CD8+ cells with increasing age has been reported (GREELEY et al., 2001). Further, a rise in the percentage numbers of CD3+ and CD8+, and a decline in CD4+ and B cells have been documented (BYRNE et al., 2000; FALDYNA et al., 2001; HEATON et al., 2002).

HEATON et al. (2002) and FALDYNA et al. (2001) found the CD4/CD8 ratio to decrease with increasing age. A recent study investigated lymphocyte subset changes during the first three months of life in puppies (TOMAN et al., 2002). Compared to adults, total lymphocyte counts remained higher in puppies at completion of the study. The CD21+ B cell percentage was persistently higher all the time, while CD3+, CD5+ and CD8+ cells increased from very low levels in newborns to almost normal adult values at 3 months. Other species in which the influence of age on circulating lymphocyte subsets has been investigated include horses and cats (ENGLISH et al., 1994; SELLON et al., 1996; MCFARLANE et al., 2001). Interestingly, in horses the CD4/CD8 ratio increases with age (MCFARLANE et al., 2000). Cats show an increase in the percentage of CD 8+ and a decrease in CD4+ and CD21+ cells. The CD4/CD8 ratio in cats has been reported to decrease with advancing age (SELLON et al., 1996;

HEATON et al., 2002), similar to humans and dogs.

2.7 Influence of gender on the peripheral blood lymphocytes in healthy dogs

An interesting feature of many diseases in humans and animals is that females are highly susceptible to autoimmune disease compared to males (ROBERTS et al., 2001).

Investigations on the influence of gender on the peripheral blood lymphocytes in healthy men and women have reported lower relative counts of CD3+ T cells and CD4+ T cells in males (BRYNAT et al., 1996; BARTLETT et al., 1998; SANTAGOSTINO et al., 1999). In contrast, SHAHGHASEMPOUR et al. (2001) found the difference between genders in CD3+ T cells to be non-significant, while finding lower CD4+ T cell numbers in men. Lower numbers of B cells (BARTLETT et al., 1998), NK cells (BRYNAT et al., 1996; SANTAGOSTINO et al., 1999) and higher CD4/CD8 ratios (CHOONG et al., 1995) in females than males have also been reported.

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In dogs, a lower relative count of T cells in the peripheral blood of males has been reported by GREELEY et al. (1996). Non-significant lower relative counts of CD3+ and CD4+ T cells and a correspondingly higher percentage of CD21+ B cells in males than females have been found by FALDYNA et al. (2001), whereas other studies did not find a significant difference in peripheral blood lymphocyte subsets between male and female dogs (FRANZ, 1996;

CULMSEE, 2000;). In cats, HOFFMANN et al. (1992) described relative counts of CD4+ T cells to be significantly higher in female when compared to male cats.

2.8 Influence of corticosteroids on the peripheral blood lymphocytes in healthy dogs Immunosuppression is the cornerstone in the management of autoimmune or immune- mediated disease in people and animals. In both human and veterinary medicine, corticosteroids are widely used alone or in combination with other immunosuppressive agents. Corticosteroids are reportedly known to affect the distribution, proliferation, differentiation and function of immunologically important cells including neutrophils, eosinophils, monocytes and lymphocytes, mainly through modulation of gene expression (MILLER, 1997; BARNES, 1998).

It has been known that corticosteroid therapy causes lymphopenia since the pioneering study of DOUGHERTY and WHITE (1944). Short-term effects of corticosteroids on lymphocyte subsets in healthy volunteers have been reported before the advent of flow cytometry and monoclonal antibodies by YU et al. (1974) and FAUCI et al. (1975, 1976).

Their results showed lymphopenia to peak 4-6 hours after a single dose of corticosteroids.

Subsequently, ten BERGE et al. (1984), using flow cytometry and monoclonal antibodies, found T cells, particularly OKT4+ (CD4+) cells to be reduced six hours after a single dose of prednisolone in normal individuals. Interestingly their results documented a disappearance of

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of mainly CD4+ cells. At 72 hours there was a significant rise in the absolute numbers of lymphocytes, T cells, CD4+, CD8+ and B cells. When comparing percentages, he found CD4+ cells to rise significantly at 72 hours while CD8+ had fallen significantly. NK cells had fallen at 72 hours in contrast to B cell numbers, which remained increased at the same point in time. In the study of UHL et al (2002), healthy volunteers were randomly assigned to receive 32 mg IV methylprednisolone either as a single dose or equally divided 12 hours apart in an experimental cross-over design. Differences in immunosuppressive effects using 24 hour effect areas where apparent, as splitting the methylprednisolone dose in two fractions resulted in a significant supression of lymphocytes compared to a single dose of the T subsets studied, CD4+ cells exhibited the strongest decline. Experiments on the short term effects of different corticosteroid preparations on circulating lymphocytes and their subsets using corticosteroid sensitive rodent models have yielded a similar pattern of early transient suppression followed by a rebound effect 24 hours later, with the quality of suppression differing among preparations (FAUCI 1976; FREY et al., 1984). If corticosteroids are given over a longer period though, changes in lymphocyte subsets appear to differ from those observed in man. FAUCI (1975), investigating the effect of long term subcutaneous cortisone acetate in guinea pigs observed a fall in lymphocytes and T cells persisting from the acute stage to seven days.

Surprisingly, this interesting and important area of research has received little attention in small animal immunology. Although neutrophilia, lymphopenia and eosinopenia among the circulating cellular components of the immune system along with changes in biochemical markers are well documented effects of corticosteroid administration in dogs, only one study investigating the effects of corticosteroids on peripheral blood lymphocyte subsets in healthy dogs has been published. In this study of RINKARDT et al. (1999), the effects on lymphocyte subsets of a long term, two week trial of oral 2mg/kg SID prednisone was compared to baseline, to a 14 day course of 2mg/kg oral SID azathioprine and to a combined, 2mg/kg oral SID prednisone and azathioprine protocol. Prednisone therapy induced a significant decline in absolute numbers of Thy-1, CD4+, CD8+ and B cells compared to baseline, while the post prednisone CD4/CD8 ratio remained unchanged. In the azathioprine treated dogs the absolute

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numbers of lymphocyte subsets remained unchanged, while the dogs in the combined protocol group exhibited significant declines in the absolute numbers of Thy-1 and CD8+

cells only. These results suggest single agent immunosuppressive therapy with corticosteroids to be most effective in dogs, while for azathioprine alone or in combination, neither immunosuppressive effect for the former, nor synergistic effects for the latter, could be demonstrated in the time frame studied.

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Materials and Methods

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3 Materials and Methods 3.1 Materials

3.1.1 Animals

The study was carried out at the Small Animal Clinic of the Hannover School of Veterinary Medicine, Germany. The study population consisted of 342 clinically healthy dogs of 6 different breeds, including mixed breed dogs (n=82), Bernese mountain dogs (n=79) Weimaraners (n=75), Flat-coated Retrievers (n=48), Rottweilers (n=36) and Beagles (n=22).

Gender, age and breed distribution of these dogs are given in tables 1, 2 and 3.

The study population for the investigation of the effect of methylprednisolone on cellular immune status consisted of five healthy Beagles. The dogs were between 12 and 13 years old (4 males and 1 female).

Table 1

Gender distribution of the clinically healthy dogs in the study.

Gender Intact Castrated Total

male 138 29 167

female 140 35 175

Total 278 64 342

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Table 2

Age distribution of the clinically healthy dogs within the study. BMD: Bernese mountain dogs; FCR: Flat-coated Retrievers; MBD: mixed breed dogs.

Age

(years) MBD BMD Weimaraner FCR Rottweiler Beagle Total

< 1 7 4 7 5 3 26

1-2 22 30 43 17 7 16 135

2-3 7 10 10 4 7 38

3-4 6 6 2 4 9 27

4-5 6 1 1 8

5-6 1 9 3 5 4 22

6-7 4 2 3 4 2 15

7-8 4 7 3 3 17

> 8 31 5 6 6 6 54

Total 82 79 75 48 36 22 342

Table 3

Breed distribution of the clinically healthy dogs within the study.

Group Breed Number of dogs %

1 Mixed breed dogs 82 24

2 Bernese mountain dogs 79 23.1

3 Weimaraner 75 21.9

4 Flat-coated Retriever 48 14

5 Rottweiler 36 10.5

6 Beagle 22 6.4

Total 342 100

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3.1.2 Antibodies

The primary and secondary monoclonal antibodies (mAb) used in this study are listed in table 4 and table 5. Optimal primary and secondary antibody dilutions were determined by serial dilutions [1:1, 1:2, 1:4, 1:8, 1:10, 1:20, 1:40: 1:80] of the respective antibody. The concentrations and dilutions of the antibodies used in this study are shown in table 6.

Table 4

Primary antibodies used for lymphocyte immunophenotyping. CD: cluster of differentiation, Ig: Immunoglobulin.

CD/antigen Clone Origin Species Isotype Conjugation canine CD3 CA17.2A12 Serotec Mouse IgG1 -

canine CD4 YKIX302.9 Serotec Rat IgG2a FITC

canine CD8a YCATE55.9 Serotec Rat IgG1 PE

canine CD45 YKIX16.13 Serotec Rat IgG2b PE

human CD14 M5E2 Pharmingen Mouse IgG2a FITC canine CD21

(B cells) CA2.1D6 Serotect Mouse IgG1 -

Table 5

Secondary antibodies used for lymphocyte immunophenotyping. CD: cluster of differentiation, Ig: Immunoglobulin.

Specificity Clone Origin Species Isotype Conjugation mouse IgG1 X36 Becton Dickinson Rat IgG1 PerCP

mouse IgG1 X36 Becton Dickinson Rat IgG1 PE

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Table 6

Basic concentrations and working dilutions of the antibodies used in the study. All antibodies were diluted in Cellwash®. CD: cluster of differentiation, Ig: immunoglobulin.

Specificity Clone Ig-Concentration Dilution

canine CD3 CA17.2A12 1mg/ml 1:20

canine CD4 YKIX 302.9 0,1 mg/ml 1:1

canine CD5 YKIX322.3 0,1 mg/ml 1:1

canine CD8a YCATE55.9 0,1mg/ml 1:1

canine CD21 CA2.1D6 1mg/ml 1:20

canine CD45 YKIX16.13 1mg/ml 1:1

human CD14 MRE2 1mg/ml 1:20

mouse IgG1 X36 1mg/ml 1:1

(27)

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3.1.3 Reagents

CellFIX, Becton Dickinson, Heidelberg CellWASH, Becton Dickinson, Heidelberg FACS Flow, Becton Dickinson, Heidelberg

FACS Lysing Solution, Becton Dickinson, Heidelberg FACS Calibur, Becton Dickinson, Heidelberg

EDTA-Blood collecting tubes, Sartedt, Nürnberg

FALCON® Test-tube12x75 mm, Becton Dickinson, Heidelberg Automate Cellcounter, Technicon H1E®, Bayer, Leverkusen Pipette, Brand, Wertheim

Pipette tips, Brand, Wertheim

Medarte®, Pharmacia GmbH, Erlangen

(28)

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3.2 Methods

3.2.1 Sample collection

Blood samples were collected by venipuncture into EDTA blood collecting tubes. A complete blood count and a hemogram were performed using an automated hematology counter (Technicon H1E®). All blood samples were stored at room temperature until analysis. None of the samples were stored longer than 24 hour until staining of cells.

3.2.2 Effect of methylprednisolone on peripheral blood lymphocyte subsets.

3.2.2.1 Experiment 1

Dogs were given methylprednisolone (at 8.30 a.m.) at a dosage of 1 mg/kg i.v., beginning at day 3 to day 6 of the study (4 treatments). During the experiment, blood samples were collected from a cephalic vein immediately before administration of methylprednisolone. In addition to lymphocyte immunophenotyping, a complete blood count (CBC), differential count and serum biochemical analysis, including alkaline phosphatase (AP), alanine aminotransferase (ALT), glutamate-dehydrogenase (GLDH) and a complete daily physical examination were performed. The protocol for methylprednisolone administration and blood collection is given in table 7.

Table 7

Protocol for blood sampling and administration of methylprednisolone in experiment 1.

I II

(29)

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3.2.2.2 Experiment 2

Four months after the first experiment, a second experiment was performed (same dogs and conditions). Dogs were given methylprednisolone (8.30 a.m.) at a dosage of 2 mg/kg i.m.

at days 3 to 7 of the study (5 treatments), at a dosage of 1 mg/kg i.m. at day 8 to day 10 (3 treatments), and at a dosage of 0.5 mg/kg i.m. at day 11 to day 13 (3 treatments). The protocol for methylprednisolone administration and blood collection is given in table 8.

Table 8

Protocol for blood sampling and administration of methylprednisolone in experiment 2.

I II III

Day 1 and 2 3-14 14-19

Blood collection 2 times per day (8.00 am and 4.00 p.m.)

Day 3,4 and 5 (2 times per day, at 8.00 am and 4.00 p.m.) Day 10 and 12 (1 time per day, at 4.00 p.m.)

Day 15 and 17 (1 time per day, at 4.00 p.m.)

Methylprednisolone - Day 3-7 (2 mg/kg i.m.) Day 8-10 (1 mg/kg i.m.)

Day 11-13 (0.5 mg/kg i.m.) -

(30)

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3.2.3 Staining of cells

For the determination of lymphocyte subsets, a two-colour immunofluorescence technique (caCD5 FITC / caCD21 PE) and a three-colour immunofluorescence technique (caCD4 FITC / caCD8a PE /caCD3 PerCP) was used, as described by CULMSEE (2000). For lymphocyte gate setting and determination of lymphocyte recovery and purity, the cells were incubated with monoclonal antibodies against CD14 and caCD45. Isotype controls were also included in each analysis (table 9).

100 µl of blood was incubated with 10 µl of the antibodies at room temperature (RT) for 15 min in the dark. The cells were washed in Cellwash® (300 g, 5 min, RT). After incubation with 10 µl of the secondary antibodies for 15 min at RT in the dark, red blood cells were lysed by adding 2 ml of FACS lysing solution10x concentrate diluted 1:10 in deionized water. The cells were incubated for 5 min at RT in the dark, centrifuged at 300 g for 5 min at RT, and the supernatant was removed. Finally, the cells were washed twice in Cellwash® (300 g, 5 min) and fixed in 250 ml CellFix® solution diluted 1:10 in deionized water.

Table 9

Monoclonal antibody combinations used in the study. CD: cluster of differentiation, PE:

phycoerythrin, ca: canine, FITC: Fluoresceinisothyocyanat, Ig: immunoglobulin, PerCP:

peridinin-chlorophyll-a

Tube Antibody 1 Antibody 2 Antibody 3

1 IgG1-FITC IgG1-PE IgG1-PerCP

2 CD14-FITC caCD45 -PE

3 caCD4-FITC caCD45 -PE caCD3-PerCP*

4 caCD5-FITC caCD21-PE*

(31)

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3.2.4 Flow cytometry measurements

Samples were analyzed within 8 hours after staining of the cells on a flow cytometer (FACScalibur®, Becton Dickinson). List mode data was acquired of 20,000 cells per tube.

For data acquisition and analysis CellQuest® software (Becton Dickinson) was used.

3.2.5 Data analysis

Lymphocyte subsets were analysed, as previously described by CULMSEE (2000).

Briefly, the intensity of side scatter (SSC) was plotted against the intensity of forward scatter (FSC) for each cell (figure 1). A gate was set on the lymphocytes (low SCC and FSC values) (R3 in figure 2). To verify the lymphocyte gate, the recovery of lymphocytes within the gate and the lymphocyte purity of the gate were determined (figure 2). The lymphocyte recovery was defined as the percentage of lymphocytes in the sample that are within the gate. The lymphocyte purity of the gate was defined as the percentage of lymphocytes (SHAPIRO, 1988).

The percentage of lymphocytes reacting with a particular antibody was determined as the number of cells within the gate (R3) in which measured fluorescence intensity was higher than in the negative control.

(32)

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neutrophils

monocytes

lymphocytes red blood cell debris

Figure 1

Cell subpopulations based on size (FSC) and granularity (SCC).

Light-scatter dot plot of leucocytes from canine peripheral blood. Red blood cells were lysed.

The different clusters of dots correspond to lymphocytes (low FSC, low SSC), granulocytes (high FSC, high SSC) and monocytes (high FSC, low SSC).

(33)

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R1 R3

R2

G1: R1 and R2 G2: R3 and R2 G3: R3

Lymphocyten-recovery: G2/G1*100= 90%

Lymphocyten-purity: G2/G3*100=96%

Tube CD14/CD45 Gate Event G1 3271 G2 3165 G3 3502

A C

Tube CD14/CD45 Gate Event

G1 3271

G2 3165

G3 3502

G1:R1 and R2 G2:R3 and R2 G3:R3

Lymphocyte-recovery: G2/G1*100 = 90%

Lymphocyte-purity: G2/G3*100 = 96%

B Figure 2

Light scatter gating technique for determining lymphocyte recovery and purity.

For determining the lymphocyte recovery and purity a relatively large light-scatter region (R) was set around the lymphocytes (R1 in figure 2 A). A second region (R2 in figure 2 B) was drawn around lymphocytes identified by fluorescence (bright canine CD45-positive, negative for CD14). Figure 2 C shows the light-scatter region drawn around the population of lymphocytes (R3) used for subsequent analysis.

G1 presents the total number of lymphocytes (R1 gated on R2). G2 presents the number of lymphocytes in the region (R3) for subsequent analysis (R2 gated on R3).

(34)

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3.2.6 Immunophenotyping data report

Results of the flow analysis were computed as the percentage of lymphocytes reacting with the particular antibody. Absolute lymphocyte subset counts were calculated as the product of the absolute lymphocyte count, derived from the hematology analyzer and the percentage of the lymphocyte subpopulations determined by flow cytometry.

3.2.7 Statistical analysis

Inter-group differences were tested by multivariate analysis. The Tamhane T2-test was used to confirm the differences. Differences with p < 0.01 were considered statistically significant.

The effect of methylprednisolone on the distribution of peripheral blood lymphocyte subsets was analyzed using ANOVA for related samples and thereafter a paired t-test. A p value < 0.05 was considered as being significant. All calculations were performed with SPSS software program.

(35)

Results

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4 Results 4.1 Validation

The results of reproducibility within a sample and between samples are shown in tables 10 and 11, respectively. The Coefficient of Variation (C.V.) of lymphocyte subset relative counts within five measurements of the same sample are between 0.44% and 3.1%, and between five samples of the same Beagle are between 0.78% and 3%.

Table 10

Intra-assay variation of the measuring method, when the sample was measured five times for testing validates instrumentation.

Parameter Mean Standard Deviation Coefficient of Variation

CD4+CD8-CD3+ 28.5 0.3 1.3 %

CD4-CD8-CD3- 36.9 1.1 3.1 %

CD3+ 89.9 0.4 0.5 %

CD5+CD21- 81.6 0.8 1 %

CD5-CD21+ 12.7 0.1 3.1 %

Table 11

Inter-assay variation of the measuring method, when five samples of the same Beagle were analyzed for testing validates methology.

Parameter Mean Standard Deviation Coefficient of Variation

CD4+CD8-CD3+ 33 0.9 2.8 %

CD4-CD8+CD3+ 32.5 0.6 2.5 %

CD3+ 91.8 1.1 1.1 %

CD5+CD21- 69.2 0.5 0.8 %

CD5-CD21+ 19 0.5 3 %

(36)

Results

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4.2 Gate purity and recovery

Mean gate purity in the study was 90% (min = 85%, max = 98%) and mean gate recovery in the study was 95% (min = 90%, max = 100%).

4.3 Lymphocyte subpopulations in peripheral blood of healthy dogs

The results of the determination of lymphocyte subsets in the peripheral blood collected from 342 healthy dogs are summarized in table 12 (relative counts) and table 13 (absolute counts).

Table 12

Relative counts of peripheral blood lymphocyte subsets of 342 clinically healthy dogs.

Parameter Median 5 th Percentile 95 th Percentile

Lymphocyte (%) 25 14 41

CD4+CD8-CD3+ (%) 38 23 52

CD4-CD8+CD3- (%) 20 11 38

CD5+CD21- (%) 74 47 86

CD5-CD21+ (%) 15 5 40

CD3+ (%) 74 50 89

CD4/CD8 1.9 0.7 3.6

(37)

Results

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Table 13

Absolute counts of peripheral blood lymphocyte subsets of 342 clinically healthy dogs.

Parameter Median 5 th Percentile 95 th Percentile

Lymphocyte (x10³/µL) 2.6 1.5 5.2

CD4+CD8-CD3+ (x10³/µL) 0.99 0.51 1.91

CD4-CD8-CD3- (x10³/µL) 0.56 0.25 1.15

CD5+CD21- (x10³/µL) 1.92 1.03 3.43

CD5-CD21+ (x10³/µL) 0.37 0.12 1.72

CD3+ (x10³/µL) 1.95 1.06 3.59

4.4 Influence of breed on lymphocyte subsets distribution

Significant interbreed differences (p < 0.01) are shown in tables 14 and 15. The relative counts of lymphocytes (figure 3) were significantly higher in Beagles and Flat-coated Retrievers than in the other four breeds (p < 0.01). The highest and the lowest absolute counts of lymphocytes (figure 4) were found in Weimaraners and mixed breed dogs, respectively.

Weimaraners showed significantly higher absolute counts of lymphocytes than Bernese mountain dogs, Flat-coated Retrievers, Rottweilers and mixed breed dogs (p < 0.01). Beagles showed significantly higher absolute counts of lymphocytes than mixed breed dogs (p <

0.01).

The highest relative counts of CD4+ T cells (figure 5) were found in Bernese mountain dogs, and the lowest relative counts of CD4+ T cells were found in Weimaraners and Beagles.

Weimaraner and Beagles showed significantly lower relative counts of CD4+ T cells than the other four breeds (p < 0.01), and Bernese mountain dogs showed significantly higher relative counts of CD4+ T cells than Rottweilers (p < 0.01). No significant differences of absolute counts of CD4+ T cells (figure 6) between breeds were found (p > 0.01).

The highest and the lowest relative counts of CD8+ T cells (figure 7) were found in Beagles and Rottweilers, respectively. Beagles and mixed breed dogs showed significantly higher relative counts of CD8+ T cells than Weimaraners and Rottweilers (p < 0.01).

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Results

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Rottweilers had significantly lower absolute counts of CD8+ T cells (Figure 8) than Weimaranes, Flat-coated Retrievers, mixed breed dogs and Beagles (p < 0.01), and Beagles showed significantly higher absolute counts of CD8+ T cells than Bernese mountain dogs, Flat-coated Retrievers and mixed breed dogs (p < 0.01).

The highest relative counts of CD3+ T-cells (figure 9) and CD5+ T cells (figure 11) were found in Beagles and Bernese mountain dogs respectively, and the lowest percentages of CD3+ T cells and CD5+ T cells were found in Weimaraners. Weimaraners showed significantly lower relative counts of CD3+ and CD5+ T cells than the other five breeds (p <

0.01). Beagles, mixed breed dogs and Bernese mountain dogs showed significantly higher relative counts of CD3+ T cells than Rottweilers (p < 0.01), and mixed breed dogs and Bernese mountain dogs showed significantly higher relative counts of CD5+ T cells than Rottweilers (p < 0.01). Beagles showed significantly higher absolute counts of CD3+ T cells (figure 10) than Rottweilers and mixed breed dogs (p < 0.01). No significant differences of absolute counts of CD5+ T cells (figure 12) between breeds were found (p > 0.01).

The highest and the lowest relative counts of CD21+ B cells were found in Weimaraners and mixed breed dogs, respectively (figure 13). The relative counts of CD21+ B cells were significantly higher in Weimaraners than in the other five breeds (p < 0.05). Flat-coated Retrievers, Rottweilers and Beagles showed higher relative counts of CD21+ B cells than Bernese mountain dogs and mixed breed dogs (p < 0.01). Weimaraners showed significantly higher absolute counts of CD21+ B-cells (figure 14) than Bernese mountain dogs, Flat-coated Retrievers, Rottweilers and mixed breed dogs (p < 0.05), and Beagles showed significantly higher absolute counts of CD21+ B cells than Bernese mountain dogs and mixed breed dogs (p < 0.01).

Weimaraners had a significantly lower CD4/CD8 ratio (figure 15) than Bernese mountain dogs (p < 0.01).

(39)

Results

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Table 14

Relative counts of lymphocyte subsets in the peripheral blood of different dogs breeds. BMD:

Bernese mountain dogs; FCR: Flat-coated Retrievers; MBD: mixed breed dogs.

Parameter BMD Weimaraners FCR Rottweilers MBD Beagles (%) (n = 79) (n = 75) (n = 48) (n = 36) (n = 82) (n = 22) Lymphocyte

median 23^c,f 24^c,f 32^a,b,d,e 24^c,f 23^c,f 38^a,b,d,e (Q1-Q3) (19-31) (20-30) (28-35) (19-28) (19-28) (33-41) CD4+ T cells

median 44^b,d,f 32â,c,d,e 39^b,f 36â,b,f 40^b,f 32â,c,d,e (Q1-Q3) (39-48) (25-37) (35-46) (32-44) (35-45) (28-34) CD8+ T cells

median 20 18^e,f 21 17^e,f 22^b,d 24^b,d

(Q1-Q3) (16-25) (15-22) (17-26) (13-21) (18-29) (22-30) CD5+ T cells

median 79^b,d 60^a,c,d,e,f 75^b 72^a,b,e 78^b,d 70^b (Q1-Q3) (73-82) (49-69) (69-78) (60-77) (72-81) (66-75) CD21+B cells

median 11^b,c,d,f 24â,c,d,e,f 16â,b,e 18â,b,e 10^b,c,d,f 18â,b,e (Q1-Q3) (8-14) (17-36) (11-20) (12-27) (6-16) (16-23) CD3+ T cells

median 78^b,d 60^a,c,d,e,f 72^b 70^a,b,e,f 78^b,d 82^b,d (Q1-Q3) (73-83) (52-68) (68-79) (58-75) (73-81) (75-89) CD4+/CD8+

median 2.2^b 1.7^a 1.9 2.2 1.9 1.4

(Q1-Q3) (1.7-3) (1.4-2) (1.3-2.5) (1.8-2.9) (1.2-2.5) (1-1.6) a Significant difference versus Bernese mountain dogs.

b Significant difference versus Weimaraners.

c Significant difference versus Flat-coated Retrievers.

d Significant difference versus Rottweilers.

e Significant difference versus mixed breed dogs f Significant difference versus Beagles

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Results

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Table 15

Absolute counts of lymphocyte subsets in the peripheral blood of different dogs breeds.

BMD: Bernese mountain dogs; FCR: Flat-coated Retrievers; MBD: mixed breed dogs.

Parameter BMD Weimaraners FCR Rottweilers MBD Beagles (x10³/µl) (n =79) (n = 75) (n = 36) (n = 48) (n = 82) (n = 22)

Lymphocyte

median 2.5^b 3.5^a,c,d,e 2.6^b 2.5^b 2.3^b,f 3.3^e (Q1-Q3) (2.0-3.1) (2.5-4.6) (2.1-3.2) (2-3.1) (1.8-2.9) (2.6-4.6)

CD4+ T cells

median 1.1 1 1 0.9 0.9 1

(Q1-Q3) (0.81-1.3) (0.8-1.4) (0.7-1.3) (0.7-1.2) (0.7-1.2) (0.8-1.4)

CD8+ T cells

median 0.5^f 0.7^d 0.6^d,f 0.4^b,c,f,e 0.6^d,f 0.8^a,c,d,e (Q1-Q3) (0.4-0.7) (0.5-0.8) (0.4-0.7) (0.3-0.5) (0.4-0.7) (0.7-1)

CD5+ T cells

Median 2 2 2 1.7 1.8 2.2

(Q1-Q3) (1.6-2.5) (1.5-2.6) (1.5-2.3) (1.4-2.1) (1.4-2.1) (1.8-3.1)

CD21+ B cells

median 0.2^b,f 0.8^a,c,d,e 0.4^b 0.4^b 0.2^b,f 0.7^a,e (Q1-Q3) (0.2-0.4) (0.5-1.3) (0.3-0.6) (0.3-0.7) (0.1-0.4) (0.4-0.8)

CD3+ T cells

median 2 1.8 2 1.7^f 1.8^f 2.6^e,d

(Q1-Q3) (1.6-2.5) (1.5-2.6) (1.6-2.4) (1.3-2.1) (1.4-2.2) (2.3-3.2)

a Significant difference versus Bernese mountain dogs.

b Significant difference versus Weimaraners.

c Significant difference versus Flat-coated Retrievers.

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Results

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60

50

40

30

20

10

0

Figure 3

Effect of breed on the relative counts of peripheral blood lymphocytes. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

Lymphocytes % Lymphocytes absolute counts

Figure 4

Effect of breed on the absolute counts of peripheral blood lymphocytes. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

8

6

4

2

0

I

25%-75%

Min – Max

− Median BMD BE MBD WM FCR RW

(n=79) (n=22) (n=82) (n=75) (n=48) (n=36) Breed

BMD BE MBD WM FCR RW (n=79) (n=22) (n=82) (n=75) (n=48) (n=36)

Breed

25%-75%

I Min – Max − Median

(42)

Results

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60

50

40

30

20

10

0

Figure 5

Effect of breed on the relative counts of peripheral blood CD4+ T cells. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

CD4+ T cells % CD4+ T cells absolute counts

3,5

3,0

2,5

2,0

1,5

1,0

,5 0,0

25%-75%

I Min – Max − Median BMD BE MBD WM FCR RW

(n=79) (n=22) (n=82) (n=75) (n=48) (n=36) Breed

(43)

Results

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50

40

30

20

10

CD8+ T cells % 0

Figure 7

1,5

1,0

,5

0,0

CD8+ T cells absolute counts

25%-75%

I Min – Max − Median BMD BE MBD WM FCR RW

(n=79) (n=22) (n=82) (n=75) (n=48) (n=36) Breed

Breed

Figure 8

Effect of breed on the absolute counts of peripheral blood CD8+ T cells. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

(44)

Results

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100

80

60

40

20

0

Figure 9

5

4

3

2

1

CD3+ T cells absolute counts 0

25%-75%

Breed

BMD BE MBD WM FCR RW

CD3+ T cells %

(45)

Results

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100

80

60

40

20

CD5+ T cells % 0

Figure 11

5

4

3

2

1

0

CD5+ T cells absolute counts

BMD BE MBD (n=79) (n=22) (n=8

25%-75%

Breed

WM FCR RW 2) (n=75) (n=48) (n=36)

Breed

25%-75%

Figure 12

Effect of breed on the absolute counts of peripheral blood CD5+ T cells. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

(46)

Results

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60

40

20

0

Figure 13

Effect of breed on the relative counts of peripheral blood CD21+ B cells. BMD: Bernese mountain dogs; BE: Beagles; MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

25%-75%

6

5

4

3

2

1

0

25%-75%

CD21+ B cells absolute counts CD21+ B cells %

Breed

(47)

Results

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8

6

4

2

CD4/CD8 ratio 0 _25%-75%

Breed Figure 15

Effect of breed on the peripheral blood CD4/CD8 ratio. Bernese mountain dogs; BE: Beagles;

MBD: mixed breed dogs; WM: Weimaraners; FCR: Flat-coated Retrievers; RW: Rottweilers.

4.5 Influence of age on lymphocyte distribution.

The effect of age on lymphocyte subset distribution in the peripheral blood of healthy dogs is shown in figures 16 to 21. For subsequent analysis of age related effects on lymphocyte subset distribution, the dogs were divided into four groups: Group I: < 6 months, Group II: >

6 months < 1 year; Group III: > 1 year < 8 years, Group IV: > 8 years, according to their age (table 16).

(48)

Results

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0 10 20 30 40 50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

years

Lymphocyte (%)

Figure 16

Effect of age on the relative counts of lymphocytes (mean ± standard deviation) in the peripheral blood of healthy dogs.

0 10 20 30 40 50 60 70

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

years

CD4+ (%)

Figure 17

Effect of age on the relative counts of CD4+ T cells (mean ± standard deviation) in the peripheral blood of healthy dogs.

(49)

Results

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0 10 20 30 40 50 60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

years

CD8+(%)

Figure 18

0 20 40 60 80 100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

years

CD3+(%)

Figure 19