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 adap-
tive 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 or-
chestrated through the lymphocyte’s specific response to individual antigens.
An important role of monocytes and macrophages is to link innate immu-
nity with adaptive immunity, through their role as antigen-presenting cells.

Leukocytes
Peripheral blood leukocytes (white blood cells) are a group of closely re-
lated 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 sen-
escent 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 ref-
erence (‘‘normal’’) range is termed leukocytosis. Causes of leukocytosis

* Corresponding author.
E-mail address: joan.carrick@sconevet.com.au (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
240 CARRICK & BEGG

include bacterial infection, viral infection, traumatic injury, burn injury,

stress, corticosteroid administration, immune-mediated diseases (eg, pur-
pura haemorrhagica), epinephrine release, abnormal excessive production
(bone marrow neoplasia), abnormal migration or inability to migrate (adhe-
sion 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 in-
fection (bacterial or viral), endotoxemia, severe injury, and failure of synthe-
sis (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 rap-
idly to the site of inflammation or infection, the neutrophil is the first cellu-
lar 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 at-
tracted to the site of inflammation by chemoattractants, including comple-
ment C5a, chemokines (interleukin [IL]-8), cytokines (tumor necrosis
factor [TNF]), leukotrienes (eg, LTB4), and microbial products [3]. To mi-
grate from the circulation, neutrophils must first marginate along the endo-
thelium 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 in-
flammation, neutrophils rapidly phagocytose particles. Phagocytosis of
pathogens is greatly enhanced by coating the organism with immunoglobu-
lin and by the presence of complement; a process referred to as opsoniza-
tion. 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 fur-
ther enhances the neutrophil’s ability to phagocytose pathogens. Phagocyto-
sis 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
 PERIPHERAL BLOOD LEUKOCYTES 241

pathogens [4]. The respiratory burst releases oxygen radicals, which,

through nuclear factor (NF)-kB, further stimulate the production of proin-
flammatory 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 antigen-
presenting 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 inflamma-
tory response adequately. Senescent neutrophils, and those that have phago-
cytosed organisms, usually undergo apoptosis [6]. These apoptotic
neutrophils are cleared by tissue macrophages, and the inflammatory re-
sponse is subsequently directed toward repair and resolution through the
production of IL-23, reduced synthesis of IL-17, and subsequent downregu-
lation of granulocyte colony-stimulating factor (G-CSF) production [7]. If
there is overwhelming infection or inflammation and monocytes or macro-
phages are unable to clear all the apoptotic neutrophils, the decaying neu-
trophils 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 antimicro-
bial properties. Many bacteria and fungi are trapped by these NETs and effec-
tively 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 crit-
ical component of effective neutrophil function.

Kinetics
Neutrophils are the most abundant peripheral leukocyte, contain an ar-
senal 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 be-
ing 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 granulopoi-
esis is regulated by cytokines, primarily G-CSF. IL-3, IL-6, and granulo-
cyte-macrophage (GM)-CSF are also capable of inducing bone marrow
production of neutrophils, however [7].
The last three stages of granulopoiesis, the metamyelocyte, band neutro-
phil, 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
242 CARRICK & BEGG

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, cy-
tokines (eg, TNF, IL-6), chemokines (eg, IL-8, MIP-2), leukotrienes, bacte-
rial products, and complement factors.
There are two pools of neutrophils in the vasculature: the circulating neu-
trophils and the marginated neutrophils. These two pools are approximately
equal in size in healthy animals; however, only the circulating pool is as-
sessed 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 macro-
phages [3,7].
Neutrophils in the circulation may temporarily adhere to vascular endothe-
lial cells and then return to the circulating pool or firmly adhere to the endo-
thelium and migrate into tissues. Margination increases rapidly when
neutrophils are stimulated by inflammatory cytokines, such as TNF and IL-
8, 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 plate-
lets. 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 sub-
sequently upregulation of b2 integrins on the neutrophil, and intracellular ad-
hesion molecule (I-CAM) 1 on the endothelial cell, which creates a firm
adhesion of the two cells. Migration of neutrophils is stimulated by chemotac-
tic 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 pro-
genitor cells in the bone marrow. The number of neutrophils present in
the circulation is a balance between demand and supply; overwhelming de-
mand may result in marked neutropenia, followed by increased numbers of
circulating neutrophils (neutrophilia) as the stored cells from the bone mar-
row enter circulation and the bone marrow upregulates production. If the
overwhelming demand is sustained, neutropenia is maintained and increas-
ing 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 acute-
phase proteins, such as fibrinogen, and serum globulins is frequently re-
quired to detect the presence of a chronic inflammatory response in the
horse [1,2].
 PERIPHERAL BLOOD LEUKOCYTES 243

Neutrophilia
An increase in the number of circulating neutrophils greater than the ref-
erence range is termed neutrophilia. Causes of neutrophilia include bacterial
or viral infection, injury, stress, corticosteroid administration, immune-
mediated diseases, abnormal migration (adhesion deficit), abnormal func-
tion (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), se-
vere 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 meta-
myelocyte, the nucleus is now prominently indented with bulbous ends
(Fig. 1). The band neutrophil has a U- or S-shaped nucleus that has a rela-
tively uniform diameter, with aggregated chromatin (Figs. 2 and 3). The ma-
ture 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 Grünwald Giemsa stain, 1000 original magnification.) (Courtesy of Peter M.
Lording, BVSc, MVetSc, Melbourne, Australia.)
244 CARRICK & BEGG

Fig. 2. (A) Band neutrophils: also note two Dohle bodies (basophilic cytoplasmic inclusions in
the cell to the left; May Grünwald 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 Grünwald Giemsa stain, 1000 orig-
inal 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 stim-
ulus, vacuolations may occur. Dohle bodies are pale blue to gray cytoplas-
mic 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 Grünwald Giemsa stain, 1000 original magnifica-
tion). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)
 PERIPHERAL BLOOD LEUKOCYTES 245

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 im-
portant 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 scav-
enger 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 se-
crete many inflammatory mediators. Similar to neutrophils, monocytes and
macrophages have the capacity to phagocytose pathogens and form a phag-
osome, which fuses with cytoplasmic lysosomes that contain microbicidal
compounds. Micro-organisms and the derived ‘‘pathogen-associated molec-
ular 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 be-
tween 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 special-
ized 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, antimi-
crobial 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 mar-
row as neutrophils, the colony forming units (CFUs-GM), which are stim-
ulated to develop into a monoblast by macrophage-derived growth factors
and cytokines, including stem cell factor, IL-3, and M-CSF. There are
246 CARRICK & BEGG

Monocytes

LPS & IFN Il-4, IL-13, corticosteroids, TGF

Type 1 Macrophage Type 2 Macrophage

Tissue destruction Tissue repair
Killing intracellular parasites Parasite encapsulation

Effector Molecules Effector Molecules

IL-12, IL-23, TNF, IL-1 (high levels) IL-12, IL-23, TNF (low levels)
IL-10 (Low levels) IL-1ra, IL-10 (high levels)
M 1 chemokines (CXCL 10), M2 chemokines (CCL22)
reactive oxygen molecules, reactive nitrogen moleclues 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 prom-

onocytes. Circulating monocytes are derived from promonocytes by matu-
ration in the bone marrow and are released immediately into the
circulation. Unlike neutrophils, there is a minimal storage pool of mono-
cytes in the bone marrow. Hence, the response to inflammation requires rep-
lication of the progenitor cells in bone marrow and is a much slower
response than the neutrophil, in which an immediate large increase is possi-
ble because of release from the bone marrow storage pool. Monocytes cir-
culate 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 nor-
mal 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 mol-
ecules 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 dif-
ferent set of chemoattractants (CCL2 and CXCL9) is responsible for migra-
tion of monocytes into inflamed tissue.
 PERIPHERAL BLOOD LEUKOCYTES 247

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 ad-
ministration, autoimmune diseases, and abnormal excess production
(bone marrow neoplasia).

Monocytopenia
A decrease in the number of circulating monocytes is termed monocyto-
penia; 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). Occasion-
ally, bilobed or trilobed nuclei are seen. The cytoplasm is usually a blue-
gray 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 sur-
face of circulating monocytes. By definition, macrophages are rarely seen in
peripheral blood smears.

Fig. 5. Monocyte (upper left) and neutrophil (lower right) (May Grünwald Giemsa stain, 1000
original magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)
248 CARRICK & BEGG

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 hyper-
sensitivity responses and are able to orchestrate the killing of helminth par-
asites by an antibody, complement, and T-lymphocyte perforin-mediated
mechanism. Eosinophils and the receptors for the potent eosinophil chemo-
kine 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 cy-
totoxic 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 bacterioci-
dal 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 hy-
persensitive ponies was much greater than adherence of eosinophils from
normal ponies.

Kinetics
The production and maturation of eosinophils in the bone marrow par-
allel 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
 PERIPHERAL BLOOD LEUKOCYTES 249

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, primar-
ily 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 eosin-
ophilic 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 refer-
ence limits is termed eosinophilia. Causes of eosinophilia include parasitism,
hypersensitivity reactions, multisystemic eosinophilic syndrome, and abnor-
mal excess production (myeloid neoplasia).

Eosinopenia
It is usual not to identify any eosinophils in an examination of a periph-
eral blood smear from a horse. Hence, a reduction in the number of circu-
lating eosinophils is difficult to interpret.

Morphology
Mature eosinophils have a segmented nucleus and prominent large pink-
orange granules in the cytoplasm and are of a similar size as neutrophils. Im-
mature 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 Grünwald Giemsa stain, 1000 original
magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)
250 CARRICK & BEGG

Function
Basophils share many functions with tissue mast cells. They are impor-
tant 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 pro-
motes 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 pro-
duction and release from the bone marrow takes 2 to 3 days.

Morphology
Basophils are characterized by their prominent dark-blue to purple cyto-
plasmic 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 pe-
ripheral circulation. They are the primary white blood cell orchestrating the
adaptive immune system. Lymphocytes are classically divided into two ma-
jor groups: T and B lymphocytes, which are delineated in the primary lym-
phoid 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 Grünwald Giemsa stain, 1000 original magnification). (Courtesy of
Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)
 PERIPHERAL BLOOD LEUKOCYTES 251

Function
The different classes of lymphocytes have defined roles as effector cells
that perform a specific function or as regulator cells that control the re-
sponses of the effector cells. Each class of lymphocyte can only be deter-
mined 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 to-
tal circulating lymphocyte population. The predominant B lymphocytes in
circulation are inactive cells that are expressing IgD and IgM on the cell sur-
face [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 immunoglob-
ulin-secreting plasma cells. It is rare for plasma cells to be found in the pe-
ripheral 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 ap-
optosis of effector T cells. These specific regulatory B cells also produce an-
tibodies that bind inflammatory soluble factors, such as complement, act as

Lymphocytes

T cells (80%) NK cells (5%) B Cells (15%)

CD 3+ CD 3- IgD+ IgM +

T cells T cells T cells

CD 8+ CD 4+ CD 8+/- CD 4-
Recognise MHC1 Recognise MHC II non MHC restricted

Cytotoxic
Cytotoxic T Helper cells
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.
252 CARRICK & BEGG

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 deter-
mined by the cell surface markers that each cell type expresses. These cell
surface markers are a reflection of the function of each cell type. Approxi-
mately 80% of circulating lymphocytes are T cells, and they are character-
ized 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 mole-
cule recognize the MHC class I–associated antigen. The CD8þ T lympho-
cytes 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–anti-
body-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 impor-
tant 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 lym-
phoid 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 activ-
ity [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
 PERIPHERAL BLOOD LEUKOCYTES 253

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 refer-
ence limits is termed lymphocytosis. Causes of lymphocytosis include excite-
ment, 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 im-
munodeficiency 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 cyto-
plasm (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 pro-
longed antigenic stimulation and are most likely T lymphocytes. These reac-
tive lymphocytes are larger cells with more obvious clumped chromatin and

Fig. 9. Lymphocyte (right) and neutrophil (left) (May Grünwald Giemsa stain, 1000 original
magnification). (Courtesy of Bruce W. Parry, BVSc, PhD, Melbourne, Australia.)
254 CARRICK & BEGG

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 de-
lineation of the B cells in response to antigenic stimulation. These cells pro-
duce 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), hemocy-
tometer, and microscope. There is some inherent error in the method; how-
ever, 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 per-
formed without the delay attributable to referral of a sample to a diagnostic
laboratory.

Automated count
There are numerous commercial automated cell counters used in veteri-
nary 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]. Im-
pedance 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 cytom-
eters and quantitative buffy coat analysis. Although the latter methods can be
reliable at normal white blood cell counts, abnormal leukocytes can be misin-
terpreted; 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
 PERIPHERAL BLOOD LEUKOCYTES 255

[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 tempera-
tures (frequently O35 C). The cells become swollen and distorted, and inter-
pretation of subtle toxic changes becomes impossible. Provision of dried
blood smears to the laboratory with the sample is critical if adequate eval-
uation 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 ob-
served 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 respon-
sible 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 gram-
negative 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 eicosa-
noids. Although monocytes and macrophages are critically important in the
development of the response to endotoxin (regulatory cells), activated neutro-
phils (effector cells) are most likely the cell responsible for most of the organ
damage [3]. This concept is supported by the observation that activated neu-
trophils 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 proinflam-
matory 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 en-
dothelial 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 in-
duce SIRS in horses. Administration of black walnut extract to horses
256 CARRICK & BEGG

produces an inflammatory model of laminitis [27–30]. Black walnut admin-

istration to horses reduces the number of circulating leukocytes, and this de-
cline 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 lami-
nar tissues [33]. Sequestration of neutrophils into the laminar dermal tissue
results in release of proinflammatory mediators that damage the endothe-
lium, causing changes in blood flow, and can activate proteases, which re-
sults 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 im-
mune system for several days by a reduction in circulating neutrophils
and monocytes and reduction of their oxidative burst capacity [34]. Strenu-
ous exercise by trained and untrained horses had no effect on the basal ex-
pression 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 num-
bers and in the concentration of myeloperoxidase (MPO) in the blood, indi-
cating degranulation of neutrophils [37]. There was significant upregulation
of leukocyte gene expression in horses that successfully completed an endur-
ance 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
 PERIPHERAL BLOOD LEUKOCYTES 257

rapidly from adult macrophages. The response of foal leukocytes compared

with those of adults to R equi infection is the subject of considerable continu-
ing research. Administration of R equi hyperimmune plasma to neonatal foals
improves opsonization of the organism and antibody-directed T-cell cytotox-
icity. Although experimental infection is not prevented, foals treated with hy-
perimmune 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 leuko-
cyte cutoff of 13  109/L has a sensitivity of 95% for detecting R equi pneumo-
nia [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 mech-
anism may provide insight into improved vaccination and treatment sched-
ules for at-risk foals.

Summary
Peripheral blood leukocytes are the key components of the immune sys-
tem. All leukocytes have regulatory and effector roles in the immune re-
sponse. 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. Un-
derstanding the regulation of leukocytes may allow more effective treatment
of many of the diseases that currently afflict horses. In addition, more de-
fined intervention to control the effects of these cells at a molecular level
may provide more effective targeted therapy of inflammatory diseases.

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