Peripheral Blood Leukocytes

Vet Clin Equine 24 (2008) 239–259

Peripheral Blood Leukocytes Joan B. Carrick, BVSc, MVSc, PhDa,*, Angela P. Begg, BVSc, PhDb a

Scone Veterinary Laboratory, Scone Veterinary Hospital, 106 Liverpool Street, Scone, NSW, Australia b Symbion Vetnostics, 6 Waterloo Road, North Ryde, NSW 2113, Australia

Peripheral blood leukocytes are an integral part of the innate and adaptive immune systems. Innate (nonspecific) immunity is provided by physical barriers: complement and the major phagocytic cells, neutrophils, and monocytes and macrophages. The adaptive immune system is primarily orchestrated through the lymphocyte’s specific response to individual antigens. An important role of monocytes and macrophages is to link innate immunity with adaptive immunity, through their role as antigen-presenting cells. Leukocytes Peripheral blood leukocytes (white blood cells) are a group of closely related cells, including neutrophils, monocytes, eosinophils, basophils, and lymphocytes. These cells continuously move throughout the body by means of the blood stream, lymphatics, and their ability to migrate through tissues. Leukocytes work together by means of a complex system of protein, lipid, and carbohydrate molecules (inflammatory mediators and their receptors) to locate and kill invading pathogens and identify and remove dead and senescent cells, foreign material, and abnormal cells. Each of the leukocytes can enhance and downregulate the responses of other leukocytes, and this highly regulated response maintains homeostasis in the face of continual challenges. Leukocytosis An increase in the circulating number of leukocytes greater than the reference (‘‘normal’’) range is termed leukocytosis. Causes of leukocytosis

* Corresponding author. E-mail address: [email protected] (J.B. Carrick). 0749-0739/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cveq.2008.05.003 vetequine.theclinics.com

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include bacterial infection, viral infection, traumatic injury, burn injury, stress, corticosteroid administration, immune-mediated diseases (eg, purpura haemorrhagica), epinephrine release, abnormal excessive production (bone marrow neoplasia), abnormal migration or inability to migrate (adhesion deficiency), and abnormal function or inability to function (eg, failure of respiratory burst or phagocytosis). Leukopenia A decrease in the number of circulating leukocytes less than reference limits is termed leukopenia. Causes of leukopenia include overwhelming infection (bacterial or viral), endotoxemia, severe injury, and failure of synthesis (bone marrow disease). Neutrophils Neutrophils are the most common nucleated cell in peripheral blood of the horse [1,2]. Typically, in the healthy animal, they comprise 50% to 70% of the total leukocyte count. As the primary phagocyte to mobilize rapidly to the site of inflammation or infection, the neutrophil is the first cellular line of defense and the key component of the innate immune system. Function Neutrophils rapidly respond to inflammation caused by infection, trauma, and chemical or physical assault. They are the first phagocytes attracted to the site of inflammation by chemoattractants, including complement C5a, chemokines (interleukin [IL]-8), cytokines (tumor necrosis factor [TNF]), leukotrienes (eg, LTB4), and microbial products [3]. To migrate from the circulation, neutrophils must first marginate along the endothelium and then firmly adhere to the endothelial cells. Neutrophils then migrate through the extracellular matrix, along the concentration gradient of the chemoattractants. These processes are facilitated by the expression of selectins and adhesion molecules on the surface of the neutrophil and the endothelial cells and by the local release of proteases. At the site of inflammation, neutrophils rapidly phagocytose particles. Phagocytosis of pathogens is greatly enhanced by coating the organism with immunoglobulin and by the presence of complement; a process referred to as opsonization. In addition, neutrophils that have been activated by cytokines (TNF and IL-6) or bacterial products (endotoxin) express increased numbers of high-affinity receptors for immunoglobulin G (IgG; CD64, CD16, and CD32), endotoxin (CD14), and the complement component C3b, which further enhances the neutrophil’s ability to phagocytose pathogens. Phagocytosis triggers a respiratory burst and fusion of the phagosome with cytoplasmic granules. The combination of the respiratory burst and the toxic components of the neutrophil’s granules is effective at killing

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pathogens [4]. The respiratory burst releases oxygen radicals, which, through nuclear factor (NF)-kB, further stimulate the production of proinflammatory mediators, including TNF, IL-1, IL-6, IL-8, and macrophage inflammatory protein (MIP)-2, to attract more neutrophils and monocytes. The recruited monocytes assist with phagocytosis and because antigenpresenting cells are capable of initiating the adaptive immune response [5]. These inflammatory responses can become self-perpetuating if appropriate control mechanisms are unable to adequately downregulate the inflammatory response adequately. Senescent neutrophils, and those that have phagocytosed organisms, usually undergo apoptosis [6]. These apoptotic neutrophils are cleared by tissue macrophages, and the inflammatory response is subsequently directed toward repair and resolution through the production of IL-23, reduced synthesis of IL-17, and subsequent downregulation of granulocyte colony-stimulating factor (G-CSF) production [7]. If there is overwhelming infection or inflammation and monocytes or macrophages are unable to clear all the apoptotic neutrophils, the decaying neutrophils then release toxic metabolites into the tissue and exacerbate the inflammatory response. A subset of activated neutrophils undergoes a recently described process of ‘‘netosis’’ [8]. Approximately 30% of activated neutrophils undergo a merging of nuclear material and the contents of phagosomes; this product forms long fibrils and is released from the neutrophil as it disintegrates. These fibers form a mesh of neutrophil extracellular traps (NETs), which have potent antimicrobial properties. Many bacteria and fungi are trapped by these NETs and effectively killed. The formation of neutrophil NETs is limited in solid tissues, such as the liver; however, in organs with loose connective tissue, such as the lung, or in body cavities, such as pleural and peritoneal spaces, these NETs are a critical component of effective neutrophil function. Kinetics Neutrophils are the most abundant peripheral leukocyte, contain an arsenal of highly toxic compounds, and have the capacity to synthesize even more toxic metabolites; hence the number in circulation is highly regulated. Neutrophils are produced in the bone marrow, with the first three stages being the myeloblast, promyelocyte, and myelocyte. These are proliferative stages. A sustained increase in the number of circulating neutrophils is the result of enhanced proliferation of these stages. Stimulation of granulopoiesis is regulated by cytokines, primarily G-CSF. IL-3, IL-6, and granulocyte-macrophage (GM)-CSF are also capable of inducing bone marrow production of neutrophils, however [7]. The last three stages of granulopoiesis, the metamyelocyte, band neutrophil, and segmented neutrophil, are maturational stages (without further cell divisions). The size of the cell decreases, the nucleus becomes smaller and indents (segments), the amount of cytoplasm increases, and the function

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of the cell improves with each maturational stage [1,2,7]. There is normally a significant pool of mature neutrophils stored in the bone marrow, which can be released in response to a variety of inflammatory stimuli. Release of neutrophils from the bone marrow is regulated by G-CSF, GM-CSF, cytokines (eg, TNF, IL-6), chemokines (eg, IL-8, MIP-2), leukotrienes, bacterial products, and complement factors. There are two pools of neutrophils in the vasculature: the circulating neutrophils and the marginated neutrophils. These two pools are approximately equal in size in healthy animals; however, only the circulating pool is assessed by venipuncture. Normally, neutrophils circulate for approximately 12 hours and then marginate and migrate into the peripheral tissue, wherein they die, undergoing regulated apoptosis and phagocytosis by tissue macrophages [3,7]. Neutrophils in the circulation may temporarily adhere to vascular endothelial cells and then return to the circulating pool or firmly adhere to the endothelium and migrate into tissues. Margination increases rapidly when neutrophils are stimulated by inflammatory cytokines, such as TNF and IL8, platelet-activating factor (PAF), or bacterial products (eg, endotoxin). These proinflammatory mediators stimulate the synthesis and expression of L-selectin on neutrophils and E- and P-selectin on endothelial cells and platelets. The primary role of selectins is to form a more secure attachment of the neutrophil to the endothelium so that migration can be initiated. There is subsequently upregulation of b2 integrins on the neutrophil, and intracellular adhesion molecule (I-CAM) 1 on the endothelial cell, which creates a firm adhesion of the two cells. Migration of neutrophils is stimulated by chemotactic factors, such as LTB4, IL-8, and the cystine-X-cystine chemokines. A sudden increase in demand for neutrophils may result in depletion of the number of circulating neutrophils (neutropenia). Many of the cytokines that initiate increased demand also induce proliferation of neutrophil progenitor cells in the bone marrow. The number of neutrophils present in the circulation is a balance between demand and supply; overwhelming demand may result in marked neutropenia, followed by increased numbers of circulating neutrophils (neutrophilia) as the stored cells from the bone marrow enter circulation and the bone marrow upregulates production. If the overwhelming demand is sustained, neutropenia is maintained and increasing numbers of immature band forms enter the circulation, producing a left shift. Sustained neutropenia with a left shift is frequently associated with a poor to grave prognosis. In contrast, sustained low-grade or chronic inflammation results in initial upregulation of bone marrow synthesis, but the equine bone marrow response rapidly equilibrates with demand and sustained neutrophilia in the presence of chronic inflammation is quite rare. Assessment of acutephase proteins, such as fibrinogen, and serum globulins is frequently required to detect the presence of a chronic inflammatory response in the horse [1,2].

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Neutrophilia An increase in the number of circulating neutrophils greater than the reference range is termed neutrophilia. Causes of neutrophilia include bacterial or viral infection, injury, stress, corticosteroid administration, immunemediated diseases, abnormal migration (adhesion deficit), abnormal function (failure of phagocytosis or respiratory burst), and abnormal excess bone marrow production (bone marrow neoplasia). Neutropenia A decrease in the number of circulating neutrophils less than reference limits is termed neutropenia. Causes of neutropenia include overwhelming infection, endotoxemia (eg, colitis, leakage from compromised bowel), severe injury, and failure of production (eg, radiation, cytotoxic drugs, bone marrow disease). Morphology The maturational stages of neutrophils are readily distinguished by the morphology of the cells. The immature proliferative stages have large round nuclei, and their azurophilic granules are quite prominent. By the time the developing neutrophil has completed proliferation and has become a metamyelocyte, the nucleus is now prominently indented with bulbous ends (Fig. 1). The band neutrophil has a U- or S-shaped nucleus that has a relatively uniform diameter, with aggregated chromatin (Figs. 2 and 3). The mature neutrophil has a nucleus that has three to five distinct lobes, and the chromatin is densely aggregated. The cytoplasm is typically pale pink with slight granulation.

Fig. 1. Metamyelocyte neutrophil: also note the azurophilic primary granules in the basophilic cytoplasm. (May Gru¨nwald Giemsa stain, 1000 original magnification.) (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

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Fig. 2. (A) Band neutrophils: also note two Dohle bodies (basophilic cytoplasmic inclusions in the cell to the left; May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Dr. P.M. Lording, Melbourne, Australia.) (B) Band neutrophils: ring form on left, whereas the cell on the right has its nucleus folded over itself (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

Toxic changes occur when the neutrophils are stimulated by bacterial or fungal byproducts or by proinflammatory mediators. The cells tend to swell slightly, the cytoplasm becomes a bluish color and, with a more severe stimulus, vacuolations may occur. Dohle bodies are pale blue to gray cytoplasmic inclusions that reflect toxic changes in a neutrophil. Occasionally, severe toxemia can result in toxic granulation, which is characterized by purple to pink granulation of the cytoplasm. Monocytes Monocytes are usually less than 10% of circulating leukocytes. These cells are the precursors of tissue macrophages. They are capable of some

Fig. 3. Three segmented neutrophils (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

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replication and undergo significant differentiation and maturation within the tissues to acquire the specific characteristic of each tissue macrophage. Function Monocytes and macrophages are primarily phagocytes. During normal homeostasis, cells of the monocyte and macrophage lineage are critically important in removing senescent and apoptotic cells. This process involves the expression of large numbers of cell surface receptors that recognize specific proteins expressed on the surface of aging cells. Receptors on the surface of macrophages that are important in recognizing apoptotic cells include scavenger receptor-A, scavenger receptor-B, CD14, and vitronectin [9]. During infection, monocytes are critically important cells in the innate immune system because of their capacity to ingest and kill microbes and secrete many inflammatory mediators. Similar to neutrophils, monocytes and macrophages have the capacity to phagocytose pathogens and form a phagosome, which fuses with cytoplasmic lysosomes that contain microbicidal compounds. Micro-organisms and the derived ‘‘pathogen-associated molecular patterns’’ interact with cell surface receptors or intracellular signals, such as peptidoglycan recognition protein, to induce synthesis of a large number of inflammatory mediators, including eicosanoids, TNF, IL-1, IL-6 and IL-8. Monocytes are also an important source of the CSFs (G-CSF, GM-CSF) and cytokines (IL-1, IL-3, and TNF) that regulate hematopoiesis. Monocytes and macrophages create an important link between the innate and acquired immune systems because they are the primary antigen-presenting cells, processing foreign substances and associating them with their major histocompatibility complex (MHC) class I or II molecules. This allows the foreign material to induce specific T-lymphocyte activation, a critical component of the acquired immune system. In response to activation, monocytes and macrophages express specialized functional patterns that fall into two primary classifications. Classically activated monocytes and macrophages are under the influence of interferon (IFN)-g and are known as M1 macrophages [10]. In contrast, macrophages that are regulated by IL-4, IL-13, and IL-10 are termed alternate-activated cells or M2 macrophages. Classically activated macrophages tend to produce proinflammatory cytokines and are involved in tissue destruction, antimicrobial activity, and disposal of tissue debris (Fig. 4). Macrophages that are alternate activated are responsible for antigen presentation and tissue and wound healing and repair [10]. Kinetics Monocytes are derived from the same progenitor cells in the bone marrow as neutrophils, the colony forming units (CFUs-GM), which are stimulated to develop into a monoblast by macrophage-derived growth factors and cytokines, including stem cell factor, IL-3, and M-CSF. There are

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Monocytes

LPS & IFN

Type 1 Macrophage Tissue destruction Killing intracellular parasites

Il-4, IL-13, corticosteroids, TGF

Type 2 Macrophage Tissue repair Parasite encapsulation

Effector Molecules

Effector Molecules

IL-12, IL-23, TNF, IL-1 (high levels) IL-10 (Low levels) M 1 chemokines (CXCL 10), reactive oxygen molecules, reactive nitrogen moleclues

IL-12, IL-23, TNF (low levels) IL-1ra, IL-10 (high levels) M2 chemokines (CCL22) Mannose, galactose receptors

Fig. 4. Under the influence of different stimuli, monocytes develop into one of two classes of macrophage, a proinflammatory form (M1) or a repair form (M2). Each class of macrophage produces a different array of cytokines to orchestrate the specific effects of the macrophages.

several replications at the monoblast stage before the development to promonocytes. Circulating monocytes are derived from promonocytes by maturation in the bone marrow and are released immediately into the circulation. Unlike neutrophils, there is a minimal storage pool of monocytes in the bone marrow. Hence, the response to inflammation requires replication of the progenitor cells in bone marrow and is a much slower response than the neutrophil, in which an immediate large increase is possible because of release from the bone marrow storage pool. Monocytes circulate in blood for 1 to 3 days. There are two separate populations of monocytes in the circulation, which can be identified by their different cell surface receptors. One group has low CX3CR1 (a chemokine) expression and high expression of CCR2 (a chemokine) and CD62L (L-selectin); these cells migrate toward inflamed tissues. The second group has high CXC3R1 expression and low CCR2 and CD63L expression; these cells migrate to normal tissue to become the long-lived resident macrophages, such as Kupffer cells, alveolar macrophages, peritoneal macrophages, microglial cells, and pericytes [9,10]. To migrate into tissues, monocytes must first adhere to the surface of the endothelium. Adhesion molecules on the surface of the monocytes and the endothelium are essential in this process. Adhesion to the endothelium in normal tissue requires the b2-integrin adhesion molecule, whereas adhesion to endothelium in inflamed tissue is regulated by intercellular adhesion molecules 1 and 2, vascular adhesion molecule 1, very late antigen 4, L-selectin, and b2-integrin adhesion molecule. Chemoattraction into healthy tissue is regulated by a specific set of chemokines (MIP-1a, CXCL14, and CX3CL1) that match the receptors expressed by the monocyte subset. A different set of chemoattractants (CCL2 and CXCL9) is responsible for migration of monocytes into inflamed tissue.

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Monocytosis An increase in the circulating monocyte number greater than reference limits is termed monocytosis. Causes of monocytosis include acute and chronic inflammation, chronic bacterial infection, stress, corticosteroid administration, autoimmune diseases, and abnormal excess production (bone marrow neoplasia). Monocytopenia A decrease in the number of circulating monocytes is termed monocytopenia; however, because monocytes are not always seen during examination of a peripheral blood smear from healthy horses, this may be a normal finding. Morphology Monocytes are the largest circulating leukocyte. They usually have an oval-shaped nucleus, typically with a small indentation (Fig. 5). Occasionally, bilobed or trilobed nuclei are seen. The cytoplasm is usually a bluegray color. Immature forms of monocytes are rarely seen in peripheral blood smears. Prolonged storage in ethylenediaminetetraacetic acid (EDTA) can result in distinct vacuoles of variable size in the cytoplasm of normal monocytes. Fresh blood samples that have vacuolated monocytes suggest activation of the cells and may be associated with systemic disease. Occasionally, small hair-like projections (pseudopodia) are seen on the surface of circulating monocytes. By definition, macrophages are rarely seen in peripheral blood smears.

Fig. 5. Monocyte (upper left) and neutrophil (lower right) (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)

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Eosinophils Eosinophils are a minor component of the peripheral blood leukocyte population, usually comprising only 0% to 3% of the total leukocyte count in a healthy horse. Function Eosinophils are typically associated with parasitic infection and hypersensitivity responses and are able to orchestrate the killing of helminth parasites by an antibody, complement, and T-lymphocyte perforin-mediated mechanism. Eosinophils and the receptors for the potent eosinophil chemokine eotaxin are present in large amounts in the equine gastrointestinal tract, and the number of eosinophils present can be correlated to the cyathostome burden [11]. Eosinophils bind to IgE and are activated by antigen-IgE complexes to release the content of their granules. These cytoplasmic granules contain major basic protein, eosinophil peroxidase, eosinophil cationic protein, and an eosinophil-derived neurotoxin. These proteins in the granules are cytotoxic to parasites and to mammalian cells; when released, they induce an inflammatory response. Eosinophils are primarily exocytotic cells rather than phagocytic cells; cytotoxic chemicals stored in the granules are released by activated eosinophils onto the surface of targeted cells or parasites. The basic proteins in the granules are also able to induce histamine release from basophils and mast cells. Although eosinophil granules contain significant amounts of peroxidase, it is distinct from the neutrophil peroxidase and is ineffective in killing bacteria. Eosinophils are much less efficient bacteriocidal cells than neutrophils. A crucial role for eosinophils in the allergic skin condition sweet itch (Queensland itch) is well defined. The potent eosinophil chemokine eotaxin has recently been identified to be present in the skin of horses with sweet itch lesions, and eosinophils are frequently present in the skin of horses with fresh lesions [12–14]. Adherence to endothelial cells by eosinophils from hypersensitive ponies was much greater than adherence of eosinophils from normal ponies. Kinetics The production and maturation of eosinophils in the bone marrow parallel that of neutrophils. The eosinophil is derived from the same myeloid stem cell line as the neutrophils and monocytes but is differentiated into an eosinophil colony-forming unit under the control of IL-5 [15]. There are immature replicating forms similar to the neutrophil, which mature in the bone marrow to become mature eosinophils. Bone marrow production of eosinophils usually takes 2 to 6 days. Eosinophils are released from the

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bone marrow under the primary control of IL-5 and are only present in the circulation for a few days; after that time, they migrate into tissues, primarily the skin, gastrointestinal tract, and lungs. Occasionally, large numbers of eosinophils accumulate in the gastrointestinal tract of the horse and can cause colic, diarrhea, or protein-losing granulomatous enteropathy. The cause of the accumulation of eosinophils is elusive. In rare cases, the eosinophilic infiltration is widespread throughout many organs and the horses can present with weight loss, diarrhea, skin lesions, and liver dysfunction [16]. Eosinophilia An increase in the number of circulating eosinophils greater than reference limits is termed eosinophilia. Causes of eosinophilia include parasitism, hypersensitivity reactions, multisystemic eosinophilic syndrome, and abnormal excess production (myeloid neoplasia). Eosinopenia It is usual not to identify any eosinophils in an examination of a peripheral blood smear from a horse. Hence, a reduction in the number of circulating eosinophils is difficult to interpret. Morphology Mature eosinophils have a segmented nucleus and prominent large pinkorange granules in the cytoplasm and are of a similar size as neutrophils. Immature forms of eosinophils are almost never seen in peripheral blood smears (Fig. 6).

Basophils Basophils are uncommon in the peripheral blood smear of the horse.

Fig. 6. Eosinophil (right) and neutrophil (left) (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)

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Function Basophils share many functions with tissue mast cells. They are important in the development of immediate and delayed hypersensitivity through release of mediators, such as histamine. Basophils can release heparin and inhibit hemostasis, but they can also be a source of kallikrein, which promotes hemostasis [17]. Kinetics Basophils are formed in the bone marrow primarily under the influence of IL-3. There is minimal storage of basophils in the bone marrow, and production and release from the bone marrow takes 2 to 3 days. Morphology Basophils are characterized by their prominent dark-blue to purple cytoplasmic granules that frequently obscure the nuclear structure; the cells are of a similar size as eosinophils (Fig. 7) [1,2].

Lymphocytes Lymphocytes comprise the second largest group of leukocytes in the peripheral circulation. They are the primary white blood cell orchestrating the adaptive immune system. Lymphocytes are classically divided into two major groups: T and B lymphocytes, which are delineated in the primary lymphoid tissues in the thymus (T cells) or the bone marrow (B cells). Unlike most of the other circulating leukocytes, T and B lymphocytes are capable of proliferation.

Fig. 7. Basophil (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)

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Function The different classes of lymphocytes have defined roles as effector cells that perform a specific function or as regulator cells that control the responses of the effector cells. Each class of lymphocyte can only be determined by identifying specific cell surface markers and receptors, which, in turn, define the cell’s function (Fig. 8). B lymphocytes are distinguished by the expression of immunoglobulin molecules on their cell surface. They make up approximately 15% of the total circulating lymphocyte population. The predominant B lymphocytes in circulation are inactive cells that are expressing IgD and IgM on the cell surface [18,19]. They require appropriate stimulation through activated T helper (Th) cells for further differentiation into initially B cells that express only one type of immunoglobulin class and then into specific immunoglobulin-secreting plasma cells. It is rare for plasma cells to be found in the peripheral circulation; most are associated with lymphatic tissue in which the T-cell–assisted development occurs [1,2]. Although B cells are primarily the effector cells for the humoral immune system, they also have roles as regulator cells [20]. Regulatory B cells can produce cytokines, such as IL-10, which restores lymphocyte Th1 and Th2 cell balance and directly inhibits the inflammatory cascade. In addition, they can produce transforming growth factor-b (TGFb), which induces apoptosis of effector T cells. These specific regulatory B cells also produce antibodies that bind inflammatory soluble factors, such as complement, act as

Lymphocytes

T cells (80%) CD 3+ T cells CD 8+ Recognise MHC1

NK cells (5%) CD 3-

T cells CD 4+ Recognise MHC II

Cytotoxic

B Cells (15%) IgD+ IgM +

T cells CD 8+/- CD 4non MHC restricted

T Helper cells

Cytotoxic Non antibody mediated

Th1 Cell mediated immunity IFN , IL-2, IL-3 Th2 Antibody production IL-4, Il-5 IL-13

Fig. 8. Lymphocytes are divided into three different subclasses, each of which performs specific functions. The T cells are further subclassified into three different groups according to each T lymphocyte’s specific function.

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antigen-presenting cells, and are able to enhance the clearance of apoptotic cells that can release damaging proinflammatory compounds and antigens. T-lymphocyte function is complex, and the different subclasses are determined by the cell surface markers that each cell type expresses. These cell surface markers are a reflection of the function of each cell type. Approximately 80% of circulating lymphocytes are T cells, and they are characterized by the expression of CD3 complex. In addition, approximately 90% of the circulating T lymphocytes express a CD4 or CD8 molecule. Cells that have a CD4 molecule have a T-cell receptor (TCR) that recognizes the MHC class II–associated antigen, whereas the cells that have a CD8 molecule recognize the MHC class I–associated antigen. The CD8þ T lymphocytes are predominantly cytotoxic effector cells, and the CD4þ T lymphocytes are helper cells for B-cell differentiation and are involved in delayed hypersensitivity responses. CD4þ T lymphocytes can be further subclassified into Th1 and Th2 cells according to the cytokines that each cell type produces. Th1 cells produce IFNg and IL-2, which are involved in cell-mediated immunity. Th2 cells produce IL-4 and IL-5, which are cytokines involved in induction of antibody production. There is another subclass of T lymphocytes that comprise approximately 10% of the circulating T lymphocytes and are not limited to recognizing an MHC class and express a different form of the TCR. These lymphocytes may be CD8þ or CD4/CD8 and are generally responsible for non–antibody-dependent cytotoxicity. The remaining 5% of circulating lymphocytes express neither CD3 nor immunoglobulins on their cell surface. There cells are referred to as natural killer cells and null cells and are important cytotoxic lymphocytes. Although this classification of T cells is useful and helps to define the role of T lymphocytes in many disease entities and in the CD4þ/CD8þ and Th1/ Th2 paradigm, it may be limiting and current concepts are continuing to evolve [21–23]. It is apparent that many recognized effector cells have important regulatory roles and that regulatory cells can have critical effector roles. Kinetics Lymphocytes in the peripheral circulation are derived from the secondary lymphoid tissues, tonsils, lymph node, spleen, bronchial-associated lymphoid tissue, and gut-associated lymphoid tissue. Lymphocytes are unique because they actively recirculate through the blood, tissues, and lymphoid tissue. They are long-lived cells and, unlike other leukocytes, are capable of replication and transformation to more mature cells with enhanced activity [18]. During circulation, lymphocytes are exposed to antigen-presenting cells that initiate development of the adaptive immune response. In addition, during circulation, cytotoxic cells are patrolling for transformed cells and can direct the cell-mediated immune response against abnormal cells. On re-entry to the lymphoid tissue, the primed T cells are exposed to many

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naive lymphocytes and the development of an appropriate immune response can be initiated. The synthesis and maturation of lymphocytes is controlled by a vast array of cytokinesdpredominantly ILs and IFN. Lymphocytosis An increase in the circulating number of lymphocytes greater than reference limits is termed lymphocytosis. Causes of lymphocytosis include excitement, exercise, abnormal excess production (lymphoid neoplasia), and, less commonly, immune stimulation. Young horses usually have higher numbers of circulating lymphocytes than adults. Lymphopenia A decrease in the circulating number of lymphocytes less than reference limits is termed lymphopenia. Causes of lymphopenia include corticosteroid administration, marked stress, viral infection, endotoxemia, overwhelming bacterial infection, and immunodeficiency diseases (eg, severe combined immunodeficiency disease [SCID], Fell pony syndrome). Morphology Mature lymphocytes are the smallest of the peripheral leukocytes. They have a relatively large dense nucleus and a small amount of pale blue cytoplasm (Fig. 9). A few larger lymphocytes, with less dense chromatin, an oval nucleus, and an increased amount of pale blue cytoplasm may also be seen. Prolonged storage in EDTA may cause swelling and distortion of the shape of the nucleus. Reactive lymphocytes are sometimes seen in peripheral blood during prolonged antigenic stimulation and are most likely T lymphocytes. These reactive lymphocytes are larger cells with more obvious clumped chromatin and

Fig. 9. Lymphocyte (right) and neutrophil (left) (May Gru¨nwald Giemsa stain, 1000 original magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)

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darker blue cytoplasm, possibly with a perinuclear pale zone. They can be difficult to distinguish from monocytes. Plasma cells are rarely seen in the circulation and represent the final delineation of the B cells in response to antigenic stimulation. These cells produce large amounts of antibody, which helps to opsonize antigens and promote phagocytosis by neutrophils and monocytes and macrophages. Assessment of peripheral leukocytes Manual count A manual white blood cell count can be readily performed using a Unopette system (Becton, Dickson & Company, Franklin Laldes, New Jersey), hemocytometer, and microscope. There is some inherent error in the method; however, it is useful as a rapid method that is available with minimal technology. Used in conjunction with evaluation of a stained blood smear, rapid assessment of the leukocyte response of a critically ill horse can be performed without the delay attributable to referral of a sample to a diagnostic laboratory. Automated count There are numerous commercial automated cell counters used in veterinary practices throughout the world. Many machines have been modified from human medicine for use with veterinary patients, and appropriate quality control is essential to ensure accurate and reliable results [1,2]. Impedance cell counters are the most commonly available automated counters used. These machines determine the number of cells that fall within a defined volume range. The leukocyte count is determined after the cells have been lysed and counts the number of particles (nuclei) present. Some machines are able to perform a differential count; however, these values should always be checked by examination of a stained blood smear. Other automated cell counter techniques include optical or laser flow cytometers and quantitative buffy coat analysis. Although the latter methods can be reliable at normal white blood cell counts, abnormal leukocytes can be misinterpreted; it is critical that appropriate quality control is maintained and that all counts are visually checked by examination of a stained blood smear. Microscopic evaluation of a stained blood smear The aim of a good blood smear is to have a region of the slide that allows adequate assessment of the morphology of the white blood cells. The region needs to have the red blood cells (RBCs) in a monolayer and at a density at which they occasionally touch each other. Placing the optimal amount of blood on the slide, spreading the blood evenly, and developing a smooth smearing technique are essential if consistent blood smears are to be made

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[24]. There are three-step commercial staining methods available (Diff Quik, Fronine, Lomb Scientific, Taren Point, NSW, Australia). The use of fresh stains is critical in creating a slide that can be readily interpreted. The most common problem when assessing blood smears in our practice laboratory is the prolonged storage of blood in EDTA at room temperatures (frequently O35 C). The cells become swollen and distorted, and interpretation of subtle toxic changes becomes impossible. Provision of dried blood smears to the laboratory with the sample is critical if adequate evaluation of the leukocyte morphology is to be made. Neutrophils and lymphocytes may agglutinate when samples are stored at less than body temperature because of cold agglutinating antibodies, thus falsely lowering total white blood cell counts. Clumping can be observed on scanning the smear. Role of leukocytes in selected equine diseases Systemic inflammatory response syndrome: endotoxemia Horses have an inflammatory system that is exquisitely primed to respond to challenge, particularly from the gram-negative bacterial cell wall product, endotoxin. The monocyte and macrophage cells are the primary cells responsible for initiating and directing the inflammatory response to endotoxin [25]. Many of the conditions that result in the largest number of critically ill horses, surgical colic, colitis, and severe pleuropneumonia have endotoxin or gramnegative bacteria in the circulation. Peripheral monocytes and tissue macrophages of the M-1 or classically activated type are designed to respond to endotoxin with the production of proinflammatory cytokines and eicosanoids. Although monocytes and macrophages are critically important in the development of the response to endotoxin (regulatory cells), activated neutrophils (effector cells) are most likely the cell responsible for most of the organ damage [3]. This concept is supported by the observation that activated neutrophils are present in the blood of all horses examined with inflammatory bowel diseases and in some of the horses with strangulating colic [26]. The morphologic activation of the neutrophils was correlated with an adverse outcome. Activated neutrophils marginate in response to endotoxin, and local release of cytokines further stimulates the cells to synthesize more proinflammatory cytokines. After exposure to endotoxin, neutrophil migration through the endothelium is delayed. The local release of proinflammatory cytokines and the contents of cytoplasmic granules from decaying neutrophils at the endothelial surface induce widespread vascular damageda hallmark of systemic inflammatory response syndrome (SIRS) [3]. Laminitis Laminitis is a condition that is closely associated with diseases that induce SIRS in horses. Administration of black walnut extract to horses

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produces an inflammatory model of laminitis [27–30]. Black walnut administration to horses reduces the number of circulating leukocytes, and this decline is greater in horses that develop clinical signs of laminitis. In addition, the leukocytes from horses that developed laminitis had a higher production of reactive oxygen species [31]. The normal dermal microvasculature has few marginated neutrophils; however, during induction of laminitis with black walnut extract, the number of neutrophils in the laminar microvasculature increases significantly [28,32]. In addition, during the prodromal and acute phases of black walnut–induced laminitis, there is an increase in the number of neutrophils and the inactive form of matrix metalloproteinase-9 in laminar tissues [33]. Sequestration of neutrophils into the laminar dermal tissue results in release of proinflammatory mediators that damage the endothelium, causing changes in blood flow, and can activate proteases, which results in the destruction of the critical supporting extracellular matrix of the hoof. This destruction leads to the severe pain, marked loss of function, and, ultimately, rotation of the pedal bone. Sequential control of monocyte and neutrophil activation may prove critically important in preventing the development of laminitis in horses with SIRS. Exercise Prolonged high-intensity exercise causes suppression of the innate immune system for several days by a reduction in circulating neutrophils and monocytes and reduction of their oxidative burst capacity [34]. Strenuous exercise by trained and untrained horses had no effect on the basal expression of IL-12, IL-4, or IFNg in peripheral blood monocytes, however [35]. In contrast, moderate exercise has minimal effects on neutrophils, phagocytosis, and oxidative metabolism in trained or untrained horses [36]. Endurance exercise induces an increase in peripheral granulocyte numbers and in the concentration of myeloperoxidase (MPO) in the blood, indicating degranulation of neutrophils [37]. There was significant upregulation of leukocyte gene expression in horses that successfully completed an endurance event. Horses that failed to complete the event had more genes in their leukocytes downregulated than the successful horses [38]. Lymphocytes from horses after submaximal exercise have reduced proliferative responses, which is associated with a high level of homocysteine in their blood [39]. Training increases the expression of Il-1b and TNFa by equine leukocytes, but has no effect on IL-2, IL-4, IL-6, or IL-10 expression, and young colts undergoing a training program have improved capacity of the neutrophils to digest foreign particles; however, other parameters, such as adherence and chemotaxis, are not altered [40,41]. Rhodococcus equi pneumonia Rhodococcus equi is an important pathogen of foals from 1 to 6 months of age. The organism is able to survive in foal macrophages but is cleared

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rapidly from adult macrophages. The response of foal leukocytes compared with those of adults to R equi infection is the subject of considerable continuing research. Administration of R equi hyperimmune plasma to neonatal foals improves opsonization of the organism and antibody-directed T-cell cytotoxicity. Although experimental infection is not prevented, foals treated with hyperimmune plasma have less severe radiographic signs and take longer to show clinical signs [42]. Foals that become clinically affected from farms where R equi is endemic have lower total white blood cell counts and lower CD4þ lymphocyte counts at 2 and 4 weeks of age. Unfortunately, these parameters are not clinically useful predictors [43]. A marked leukocytosis is characteristic of infection with R equi, and serial evaluation of total white blood cell count is a useful predictor of foals that have early clinical disease. A peripheral leukocyte cutoff of 13  109/L has a sensitivity of 95% for detecting R equi pneumonia [44]. Virulent R equi is cleared from the lungs of adult horses in association with increased production of IFNg by CD8þ and CD4þ lymphocytes [45]. Foals are unable to produce IFNg at birth; however, their lymphocytes’ ability to produce the cytokine, in response to R equi infection, increases during the first 6 months of life [46,47]. Adult equine cytotoxic lymphocytes kill macrophages that are infected with R equi in a non-MHC class I–restricted fashion, which indicates novel antigen processing and presentation [48]. Further elucidation of this mechanism may provide insight into improved vaccination and treatment schedules for at-risk foals. Summary Peripheral blood leukocytes are the key components of the immune system. All leukocytes have regulatory and effector roles in the immune response. The molecular control of the function of each of the white blood cells has been the subject of intense research for longer than 20 years. Understanding the regulation of leukocytes may allow more effective treatment of many of the diseases that currently afflict horses. In addition, more defined intervention to control the effects of these cells at a molecular level may provide more effective targeted therapy of inflammatory diseases. References [1] Latimer KS, Prasse KW. Leukocytes. In: Latimer KS, Mahaffey EA, Prasse KW, editors. Duncan and Prasse’s veterinary laboratory medicine clinical pathology. 4th edition. Ames (IA): Iowa State Press; 2003. p. 46–79. [2] Stockham SL, Scott MA. Leukocytes. Fundamentals of veterinary clinical pathology. 1st edition. Ames (IA): Iowa State Press; 2002. p. 49–83. [3] Brown KA, Brain SD, Pearson JD, et al. Neutrophils in development of multiple organ failure. Lancet 2006;368:157–69. [4] Urban CF, Lourido S, Zychlinsky A. How do microbes evade neutrophil killing. Cell Microbiol 2006;8(11):1687–96.

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[5] Fialkow L, Wang Y, Downey GP. Reactive oxygen and nitrogen species as signalling molecules regulating neutrophils function. Free Radic Biol Med 2007;42:153–64. [6] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2005;6:1191–7. [7] Christopher MJ, Link DC. Regulation of neutrophil homeostasis. Curr Opin Hematol 2007; 14:3–8. [8] Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 2007;5:577–82. [9] Cavaillon J-M, Minou A-C. Monocytes/macrophages and sepsis. Crit Care Med 2005; 33(Suppl 12):S506–9. [10] Mantavani A, Sica A, Locati M. New vistas on macrophage differentiation and activation. Eur J Immunol 2007;37:14–6. [11] Collobert-Laugier C, Hoste H, Sevin C, et al. Mast cell and eosinophil mucosal responses in the large intestine of horses naturally infected with cyathostomes. Vet Parasitol 2002;107: 251–64. [12] Benarafa C, Collins ME, Hamblin AS, et al. Role of the chemokine eotaxin in the pathogenesis of equine sweet itch. Vet Rec 2002;151(23):691–3. [13] Hubert J. Equine eosinophilsdwhy do they migrate? Vet J 2006;171(3):389–92. [14] Weston MC, Cunningham FM, Collins ME. Distribution of CCR3 mRNA expression in horse tissues. Vet Immunol Immunopathol 2006;114(3–4):238–46. [15] Sampson. The role of eosinophils and neutrophils in inflammation. Clin Exp Allergy 2000; 30(Suppl 1):22–7. [16] Nimmo Wilkie JS, Yager JA, Nation PN, et al. Chronic eosinophilic dermatitis: a manifestation of a multisystemic epitheliotropic disease in 5 horses. Vet Pathol 1985;22:297–305. [17] Holgate ST. The role of mast cells and basophils in inflammation. Clin Exp Allergy 2000; 30(Suppl 1):28–32. [18] Lunn DP, Horohov DW. Equine immunology. In: Reed SM, Bayly WM, Sellon DC, editors. Equine internal medicine. 2nd edition. St Louis (MO): Saunders; 2004. p. 1–28. [19] Stobo JD. Lymphocytes: development and function. In: Gallin JI, Goldstein IM, Snyderman R, editors. Inflammation: basic principles and clinical correlates. New York: Raven Press Ltd; 1988. [20] Mizoguchi A, Bhan AK. A case for regulatory B cells. J Immunol 2006;176:705–10. [21] Chaouat G, Ledee-Bataile N, Dubanchet S, et al. Th1/Th2 paradigm in pregnancy: paradigm lost? Int Arch Allergy Immunol 2004;134:93–119. [22] Ronacarlo MG, Battaglia M. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol 2007;7(8):585–98. [23] Scharnagl NC, Klade CS. Experimental discovery of T-cell epitopes: combining the best of classical and contemporary approaches. Expert Rev Vaccines 2007;6(4):605–15. [24] Latimer KS, Rakich P. Peripheral blood smears. In: Cowell RL, Tyler RD, editors. Diagnostic cytology and hematology of the horse. 2nd edition. St Louis (MO): Mosby Inc; 2002. [25] Moore JN, Barton MH. Treatment of endotoxemia. Vet Clin North Am Equine Pract 2003; 19(3):681–95. [26] Weiss DJ, Evanson OA. Evaluation of activated neutrophils in the blood of horses with colic. Am J Vet Res 2003;64(11):1364–8. [27] Belknap JK, Giguere S, Pettigrew A, et al. Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response. Equine Vet J 2007;39(1):42–7. [28] Black SJ, Lunn DP, Yin C, et al. Leukocyte emigration in the early stages of laminitis. Vet Immunol Immunopathol 2006;109(1–2):161–6. [29] Waguespack RW, Cochran A, Belknap JK. Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses. Am J Vet Res 2004; 65(12):1724–9.

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