Chapter 5

Markers of Inflammation
Dori R. Germolec, Kelly A. Shipkowski, Rachel P. Frawley,
and Ellen Evans

Abstract
Inflammation is a complex and necessary component of the response to biological, chemical, or physical
stimuli, and the cellular and molecular events that initiate and regulate the interactions between the various
players in the inflammatory process remain a source of ongoing investigation. In the acute phase of the
inflammatory response, cells of the immune system migrate to the site of injury in a carefully orchestrated
sequence of events that is facilitated by soluble mediators such as cytokines, chemokines, and acute-phase
proteins. Depending on the degree of injury, this acute phase may be sufficient to resolve the damage and
initiate healing processes. Persistent inflammation, either as a result of prolonged exposure to stimulation
or an inappropriate reaction against self-molecules, can lead to the chronic phase, in which tissue damage
and fibrosis can occur. Chronic inflammation has been reported to contribute to numerous diseases,
including arthritis, asthma, atherosclerosis, autoimmune diseases, diabetes, and cancer, and to conditions
of aging. Hematology and clinical chemistry data from standard toxicology studies can provide an initial
indication of the presence and sometimes the location of inflammation. These data may suggest more
specific immune function assays that are necessary to determine the presence and/or mechanism(s) of
immunomodulation. Although changes in hematology dynamics, acute-phase proteins, complement fac-
tors, and cytokines are common to virtually all inflammatory conditions, and can be measured by a variety
of techniques, individual biomarkers have yet to be strongly associated with specific pathologic events.
Thus, although sensitive indicators of inflammation, these factors generally lack the specificity to identify
the offending cause. The profile seen in a given inflammatory condition is dependent on the severity, chro-
nicity, and mechanisms involved in the inflammatory process, as well as the species and the capacity of the
individual’s immune system to respond and adapt.

Key words Acute-phase proteins, Basophil, Chemokine, Clinical pathology, Complement, Cytokine,
Eosinophil, Hematology, Inflammation, Lymphocyte, Macrophage, Monocyte, Neutrophil, Platelet

1  Introduction

Inflammation is a complex and necessary component of an organ-

ism’s response to biological, chemical, and/or physical stimuli.
Inflammation is generally described as consisting of separate acute
and chronic phases, though there is overlap between these pro-
cesses. In the acute phase, leukocytes, primarily granulocytes,
migrate along a chemotactic gradient to the site of injury in a

Jamie C. DeWitt et al. (eds.), Immunotoxicity Testing: Methods and Protocols, Methods in Molecular Biology, vol. 1803,
https://doi.org/10.1007/978-1-4939-8549-4_5, © Springer Science+Business Media, LLC, part of Springer Nature 2018

57
58 Dori R. Germolec et al.

c­arefully orchestrated effort that is mediated by cytokines and

acute-­phase proteins, with the objective of removing the inflamma-
tory stimulus (e.g., infectious agent, foreign material) or cells dam-
aged by injury and initiate healing. Depending on the degree of
injury, this acute cellular phase may be sufficient to resolve any
damage. Persistent inflammation, as a result of either prolonged
exposure to inflammatory stimuli or an inappropriate reaction to
self-­molecules, can lead to the chronic phase, in which the active
immune cell populations shift to include a mononuclear pheno-
type, and tissue damage and fibrosis can occur. Chronic inflamma-
tion is reported to contribute to numerous diseases, including
arthritis, asthma, atherosclerosis, autoimmune diseases, diabetes,
and cancer, and to conditions of aging. The inflammatory process
involves multiple physiological systems, with the immune system
playing a central role [1–3]. Detailed information on the specific
cells, cell surface molecules, and soluble mediators of the inflam-
matory response is beyond the scope of this overview, and the
reader is referred to chapters which cover specific aspects of the
immune response or the topic-specific reviews cited below for
additional details.

2  General Considerations from Standard Toxicology Studies

Hematology data (including erythrocyte parameters, platelet

count, total number of leukocytes, and leukocyte differentials and
morphology), coagulation (clotting times, fibrinogen), and clinical
chemistry data (total protein, albumin and globulin, liver enzymes,
renal parameters, electrolytes, bilirubin) are included in standard
toxicology studies. These clinical pathology data can provide an
initial indication of the presence and sometimes the location of
inflammation in the absence of specific data on immune tissues.
Due to the high inter- and intra-animal variability in non-rodent
species, pretest samples should be collected for non-rodent studies,
so that experimental data can be interpreted in comparison to a
baseline; for all species, data should be compared with age-matched
concurrent and historical controls. Hematology and serum chem-
istry may provide information on both innate and acquired immu-
nity, and, in addition to basic information on immune cells, these
endpoints provide baseline information on other organ systems
that may affect or be affected by the immune system. For example,
changes in erythrocyte parameters or leukocyte total number and/
or differential (lymphocytes, neutrophils, monocytes, basophils,
and eosinophils) may indicate altered bone marrow function and
the potential for decreased production of immune cells or precur-
sors, and decreases in globulins may signal decreased antibody syn-
thesis, particularly if the albumin/globulin ratio is increased.
Increased fibrinogen may suggest an inflammatory process, even in
 Markers of Inflammation 59

the absence of an inflammatory leukogram. It is important that

these data be considered along with other available information,
such as clinical observations and histopathology, in order to distin-
guish those changes that represent direct effects of a chemical
agent on the immune system (such as a shift in leukocyte popula-
tions as a result of destruction of bone marrow progenitors or lym-
phocytes) from those that may be a secondary consequence of
immune system perturbation (such as a shift in leukocyte popula-
tions as a result of infection). The clinical pathology data may sug-
gest more specific immune function assays that are necessary to
determine the existence or mechanism(s) of immunomodulation;
however, these data alone are not always reliable predictors of
immunotoxicity. For example, circulating leukocyte numbers may
be within normal values even when there are extreme changes in
immune function, such as observed in chronic, well-established
infection and in some children with primary immunodeficiencies.
Conversely, effects on leukocyte trafficking unrelated to the
immune system may affect circulating numbers of individual white
blood cell types.

3  Cells of the Inflammatory Response

In the acute phase of inflammation, platelets and granulocytic cells

such as basophils, mast cells, neutrophils, and eosinophils are acti-
vated and in turn produce and release a number of soluble media-
tors that stimulate and regulate the inflammatory response.
Neutrophils, which are sometimes referred to as polymorphonu-
clear neutrophils (PMNs) because of the lobulated nature of their
nuclei, are the primary cellular mediators of the acute inflamma-
tory response. Their granules contain a variety of enzymes, pep-
tides, and proteins and can also rapidly release reactive oxygen
species (respiratory burst). These serve to destroy and digest
organisms and foreign material following phagocytosis, but may
also be released and damage host tissues at the inflammatory site.
Measurement of some neutrophil products, notably myeloperoxi-
dase, may be used to assess severity of inflammation [4]. Neutrophils
migrate from the blood to the site of injury as a result of vasodila-
tion and the increased vascular permeability following basophil or
mast cell degranulation, complement activation, or release of pros-
taglandins and leukotrienes, which may also be reflected as an
increase of circulating neutrophils (Fig. 1).
However, there are numerous causes of increased numbers of
circulating neutrophils (neutrophilia), and some of these may not
directly relate to immune status, which underscores the need to
integrate all of the data from a toxicology study rather than
­assessing individual components separately. Two examples of neu-
trophil trafficking effects which are not directly immune system-
60 Dori R. Germolec et al.

Fig. 1 Mediators in the process of inflammation. This figure summarizes the roles of the various mediators
important in the process of inflammation from the acute to chronic phase. Abbreviations: PGE prostaglandin E,
VIP vasoactive intestinal polypeptide, LTB leukotriene B, LTD leukotriene D, PAF platelet-activating factor, IL
interleukin, CXCL chemokine (C-X-C motif) ligand, CCL CC chemokine ligand, IFN interferon, MCP monocyte
chemoattractant protein, TGF transforming growth factor, TNF tumor necrosis factor

related include excitement and stress. Excitement with epinephrine

release results in mobilization of mature neutrophils from the bone
marrow as well as from marginal (noncirculating) pools (demar-
gination); stress and its resultant corticosteroid release lead to an
increased release from bone marrow and decreased migration to
tissues. In both cases, an increase in circulating mature neutrophils
is seen. In contrast, neutrophilia as a consequence of inflammation
is typically characterized by a shift toward immature cell types
(called a “left shift”) with increased numbers of band cells or earlier
neutrophil stages (myelocytes, promyelocytes; ring forms in
rodents), as the bone marrow depletes its reserve of mature neu-
trophils and begins releasing immature cells into circulation to
meet the demands of the site(s) of inflammation. It should be
noted that immature forms are less likely to be seen in chronic,
established infections. Specific morphologic changes such as Döhle
bodies, increased cytoplasmic basophilia, toxic granulation, and
increased vacuolation (known collectively as “toxic change”) may
be seen in any situation of accelerated myelopoiesis in the bone
marrow. The term “toxic change” is somewhat of a misnomer, in
that “toxicity” (either from a drug, chemical, or bacterial toxin) is
not necessary to bring about these morphologic changes.
Basophils and mast cells contain cytoplasmic granules that
serve as reservoirs for soluble mediators that function in many
aspects of the inflammatory response. Early-phase reactants
 Markers of Inflammation 61

released from mast cells such as the products of arachidonic acid

metabolism (prostaglandins and leukotrienes) and histamine medi-
ate the vasodilation and increased vascular permeability character-
istic of the acute vascular response. The secretion of
platelet-activating factor (PAF) by mast cells also increases vascular
permeability and, at the same time, stimulates the release of inflam-
matory mediators from platelets, resulting in the activation of neu-
trophils. Other enzymes released from mast cells that play significant
roles in tissue damage and repair include B-glucuronidase, amyloi-
dase, and chymase.
While basophil counts are routinely included in leukocyte dif-
ferentials, their low numbers (<1% of the total leukocytes in health)
make them the most difficult of the leukocytes to enumerate accu-
rately by manual differential. Unfortunately, the determination of
basophil counts by automated differential instruments is challeng-
ing as well. The size and staining characteristics of basophils can
overlap with other cell types, making it difficult for instruments to
categorize them; it has been demonstrated that automated instru-
ments tend to underestimate the counts during true basophilia [5].
Furthermore, increases in basophils are rarely significant enough to
appreciably affect the total leukocyte count. Manually scanning
peripheral blood smears may be useful to identify cases in which
the instrument underestimates basophil numbers. It has been sug-
gested that circulating numbers of basophils may be reduced in
conditions of chronic inflammation due to active recruitment of
the cells to the site of injury [6]; however, since reference intervals
for basophils generally start at 0 cells/μL, basopenia is usually not
a practical entity for diagnostic purposes. While circulating baso-
phil numbers are low in all species, they are so rare in the peripheral
blood of mice that it was once thought that basophils did not exist
in the mouse; however, more recent literature suggests that mice
do in fact possess the cells [7, 8].
Eosinophils are the predominant inflammatory cells associated
with hypersensitivity responses and clearance of parasitic infections.
Eosinophils are recruited to the site of inflammation by a number
of factors including interleukin (IL)-5, IL-2, IL-16, histamine,
neutrophil peptides, and some complement proteins. Activation of
eosinophils results in the release of eosinophil peroxidase, major
basic protein (MBP), eosinophil-derived neurotoxin, and eosino-
phil cationic proteins (ECP). In respiratory hypersensitivity
responses, these mediators induce rapid vasoconstriction followed
by increased vascular permeability and pulmonary edema. MBP
and ECP are highly basic proteins that degrade nearby cells such as
the tracheal epithelium. Automated hematology analyzers may
vary in their ability to accurately identify eosinophils across species
due to interspecies variability in granulation. However, changes in
eosinophil numbers are easy to identify in manual leukocyte dif-
ferentials, as their cytoplasmic granules have a high affinity for
62 Dori R. Germolec et al.

acidic dyes that results in a characteristic pink/red staining. In cer-

tain allergic conditions, such as asthma and rhinitis, the numbers of
circulating eosinophils may be positively correlated to disease
severity [9]. Eosinophilia may also be present during infection with
helminthic parasites. However, eosinophilic inflammatory
responses in peripheral tissues may not always result in increased
eosinophil numbers in peripheral blood [10]. MBP and ECP,
which can be evaluated in serum, sputum, and pulmonary lavage
fluid, are becoming important clinical markers for eosinophil acti-
vation and allergic disease.
Platelets are anucleate circulating cell fragments consisting of
membrane-bound cytoplasm derived from megakaryocytes. They
are rapidly recruited to sites of vascular compromise, injury, and
infection and are locally activated. The primary function of plate-
lets is to facilitate clot formation and prevent leakage from dam-
aged blood vessels. Platelets work in concert with proteins of the
coagulation system such as fibrinogen and vitronectin at sites of
vascular damage. Although they are highly differentiated for
homeostasis, platelets also have inflammatory and antimicrobial
functions and thereby link the coagulation cascade, the inflam-
matory process, and the immune system [11]. Examples of their
inflammatory functions include the release of reactive oxygen
species, which may contribute to tissue damage, and production
of mediators such as heparin and serotonin, which promote the
vasodilatory status of the acute vascular response. While periph-
eral blood platelet counts and morphology may be affected by, or
platelets may participate in, inflammatory processes, assessment
of platelet parameters alone is not helpful in identifying the pres-
ence of, or characterizing the nature of, inflammation. A number
of therapeutic agents (e.g., procainamide, sulfamethoxazole, gold
salts) can induce immune-mediated thrombocytopenias and/or
aplastic anemias (which, despite the term “anemia,” are charac-
terized by a lack of production of all major bone marrow
lineages: erythrocytes, platelets, and granulocytic cells). Immune-
mediated thrombocytopenias are generally regenerative in nature;
that is, mean platelet volume (MPV) is increased and large plate-
lets are noted on blood smears, indicating accelerated release of
platelets by megakaryocytes, which are in turn increased in num-
ber in the bone marrow. However, if the target of toxicity or an
immune-­mediated process is an earlier progenitor cell in the bone
marrow, MPV may be normal and megakaryocytes may be
reduced in number. Thrombocytosis (an increased number of
platelets in peripheral blood) may manifest as a primary response
to hemorrhage, acute inflammation, or infections or may develop
as a secondary result of some chronic inflammatory conditions
such as rheumatoid arthritis, or during liver regeneration follow-
ing hepatotoxicity or hepatectomy [12]. Conversely, tissue dam-
age and/or the inflammatory process itself may trigger
 Markers of Inflammation 63

disseminated intravascular coagulation (DIC) which results in the

depletion of platelets due to consumption as thrombi are formed
throughout the organism. DIC is characterized by decreased
platelet counts and increased MPV.  It has also been suggested
that the increased cardiovascular mortality associated with high
levels of air pollution may be linked to toxicant-induced throm-
bocytosis that initiates embolism formation [13].
Lymphocytic infiltration is often a prominent feature in chronic
inflammation. T and B lymphocytes may act as specific effectors of
cytotoxicity or secrete antibodies or cytokines that participate in
tissue damage or inflammatory cell recruitment. Innate lymphoid
cells, which lack lymphocyte antigen receptors, also secrete cyto-
kines which combat infection and contribute to inflammatory dis-
ease in a nonspecific fashion [14]. Lymphocyte responses, both
systemic and local, may have no impact on the numbers of circulat-
ing total lymphocytes or subsets, and similarly, changes in the
numbers of circulating lymphocytes are not necessarily indicative
of immune stimulation or inflammation; such changes may instead
be the result of physiologic responses mediated by epinephrine
(lymphocytosis likely due to diminished homing to peripheral lym-
phoid tissue), changes in cytokine secretion, or changes in lympho-
cyte homing and trafficking, such as that occurs in stress responses
mediated by corticosteroids (lymphopenia due to increased hom-
ing to lymphoid tissue or lysis) [15]. Lymphocytosis can occur
with lymphocytic neoplasia. It is occasionally seen with chronic
infections (e.g., Rickettsial diseases, fungal infections, etc.) or other
chronic antigenic stimulation, and in these cases, morphologically
reactive lymphocytes may be seen in peripheral blood. For most
species, lymphocytosis is not a typical component of the inflamma-
tory leukogram; lymphocytes tend to be more concentrated in
lymph nodes or sites of inflammation. In fact, stress-induced lym-
phopenia is a more typical component of inflammation. A notable
exception is the rat, where lymphocytosis is not uncommon in
inflammatory responses [7].
Macrophages tend to accumulate at the site of injury following
lymphocytic infiltration and may also be the primary response to
chronic diseases such as mycobacterial infections. They serve as
antigen-presenting cells and also help drive and perpetuate the
immune response by releasing a variety of lymphocyte-stimulating
pro-inflammatory cytokines and chemokines that play a predomi-
nant role in the regulation and resolution of the inflammatory
response. Lysosomal enzymes contained within macrophages
­participate in the phagocytic degradation of foreign materials and
inflammatory mediators. Resident macrophages in tissues such as
the lung and liver are capable of reproducing to increase inflamma-
tory cell numbers. In addition, monocytes may be recruited to
inflammatory sites from the bone marrow and continue to differ-
entiate into macrophages (Fig.  1). Monocytes and macrophages
64 Dori R. Germolec et al.

play a major role in the removal of dead or abnormal cells, and

thus, increased numbers of these cells may be indicative of inflam-
mation or tissue necrosis. Macrophages are rarely present in periph-
eral blood, but monocytes on their way to inflammatory sites may
display some characteristics of activation such as upregulation of
specific cell surface markers. Monocytosis (increases of monocytes
in peripheral blood) can occur with both acute and chronic inflam-
mation. Monocytosis may also be present as part of the
corticosteroid-­mediated stress response, but this is a less consistent
finding than is lymphopenia.
While not considered mediators or primary participants in
inflammation, red blood cells can be affected by and reflective of
inflammatory processes. Red blood cells are highly susceptible to
immune-mediated and other processes that accelerate red blood
cell destruction. The immune-mediated process and potentially
the red blood cell destruction itself may trigger an inflammatory
response; therefore, inflammatory leukograms are very common in
immune-mediated anemias and are sometimes seen in hemolytic
anemias which are not immune mediated. Hemolytic anemias are
typically very regenerative in nature, as indicated by reticulocytosis,
polychromasia, and anisocytosis. Inflammation can also lead to red
blood cell loss if significant hemorrhage occurs, either as part of
the inflammatory process or as part of DIC. This anemia may or
may not appear regenerative, depending on whether or not the
bone marrow has had time to respond (usually 2–5 days).
The most common red blood cell finding in chronic inflamma-
tion is a non-regenerative, mild-to-moderate decrease in red blood
cells termed “anemia of chronic disease (ACD)” or sometimes
“anemia of inflammatory disease” [16]. The major purpose of
ACD seems to be limiting iron availability, which is beneficial in
inflammation by reducing the potential for oxidation and free radi-
cal formation and limiting iron-dependent bacterial growth.
Multiple inflammatory cytokines are thought to play a role in
ACD, including IL-1β and tumor necrosis factor (TNF)-α, both of
which reduce erythropoietin release by the kidneys. TNF-α is also
thought to contribute to anemia via enhancement of erythropha-
gocytosis by macrophages, and along with IL-1β and interferon
(IFN)-γ, it directly decreases erythroid progenitor proliferation in
the bone marrow. In addition, IL-6 increases hepcidin production
by the liver; which in turn inhibits ferroportin-mediated release of
iron stores from macrophages and absorption of iron into
­circulation from the intestine, resulting in diminished erythropoi-
esis due to the lack of iron availability [17, 18]. Because ACD can
mimic iron deficiency anemias, serum ferritin, an acute-phase pro-
tein involved in the storage of iron, may be measured in some spe-
cies. With iron deficiency, serum ferritin concentrations are typically
decreased, whereas in ACD serum ferritin concentrations are nor-
mal or increased.
 Markers of Inflammation 65

In summary, the classic peripheral blood profile suggestive of

inflammation is dominated by neutrophilia or neutropenia with a
left shift, and monocytosis is frequently present [19]. Lymphopenia
is a common finding in inflammation in non-rodent species, pri-
marily due to secondary stress, whereas lymphocytosis is frequently
present in rodents. In addition, a non-regenerative, mild-to-­
moderate decrease in red blood cell parameters may be seen. Serum
chemistry and coagulation profiles may further support diagnosis
of inflammation through findings such as increased immunoglobu-
lins, changes in acute-phase proteins (APPs), and increased fibrino-
gen. Hematology data represent a snapshot of a very dynamic
process; therefore, a classic inflammatory profile is not always
observed and specific parameters affected are dependent on spe-
cies, mechanisms, severity, chronicity, and capacity of the immune
system to respond and adapt.

4  Cell Surface Receptors and Adhesion Molecules

Adhesion molecules control the interactions between leukocytes

and endothelial cells during the inflammatory response. These cell-­
to-­cell interactions are the result of a complex cascade of events
stimulated by the upregulation of cell surface ligands early in
inflammation. Once inflammatory cells have arrived at the site of
injury, further interactions with the endothelium, other parenchy-
mal cells, immune cells, and extracellular matrix proteins perpetu-
ate the response, leading to resolution or chronic inflammation.
Several families of adhesion molecules participate in this cascade of
events including the selectins, integrins, vascular addressins, and
lymphocyte homing receptors [20–23].

4.1  Selectins Selectins are cell surface molecules that are expressed on leukocytes
(L-selectin), endothelial cells (P-selectin, E-selectin), and platelets
(P-selectin). Selectins share a similar structure and bind to the
sialyl-Lewis X (sLEx) tetrasaccharide element found on the cell sur-
face; the predominant selecting ligand is P-selectin glycoprotein
ligand-1 (PSGL-1) [20, 21]. L-selectin initiates the interaction
between leukocytes and endothelial cells via binding to mucin-like
molecules called addressins on the vascular endothelium and
­functions as a homing receptor for lymphocytes. P-selectin is local-
ized to membrane-bound granules in endothelial cells and alpha
granules in platelets and is rapidly expressed following mast cell
degranulation. P-selectin facilitates the interactions between
PMNs, platelets, and endothelial cells and is important in both clot
formation and degradation. E-selectin expression facilitates the
binding of leukocytes at sites of inflammation and is upregulated in
the acute phase of the inflammatory response by cytokines and
bacterial cell wall products such as lipopolysaccharide (LPS) [21,
66 Dori R. Germolec et al.

23]. Acting in a tissue-specific manner, selectins mediate the cap-

ture/tethering of immune cells to the endothelium of vessel walls.
This is followed by a coordinated sequence of binding and release
between selectins and cell surface receptors to facilitate rolling to
the site of inflammation [22]. Selectins and their ligands are upreg-
ulated in many inflammatory and autoimmune diseases, e.g., pso-
riasis, inflammatory bowel disease (IBD), rheumatoid arthritis
(RA), and systemic lupus erythematosus (SLE) [20]. Selectin
ligands are also upregulated in many cancers, especially carcino-
mas, where they are associated with tumor metastasis and poor
disease prognosis [24].

4.2  Integrins The integrins are a widely expressed family of molecules that medi-
ate cell-cell interaction and interactions between cells and the
extracellular matrix and facilitate the migration of leukocytes
through the vasculature to sites of injury and inflammation [22,
25]. Integrins contain an extracellular domain that engages other
cell adhesion molecules such as intercellular adhesion molecule-1
(ICAM-1), or adhesive ligands such as fibrinogen and LPS, and a
cytoplasmic domain that interacts with intracellular proteins.
Integrins are distinguished by their ability to shift between differ-
ent conformational states, and their ability to transduce signals
bidirectionally across cell membranes. The conformational state is
critical in determining binding to ligands and activation level, as in
the inactive state, integrins are unable to bind ligand [22]. Both
selectin- and cytokine/chemokine-mediated pathways can induce
integrin activation. When fully activated, integrins mediate firm
adhesion of immune cells to receptors on the endothelium, fol-
lowed by post-arrest modifications necessary for transmigration of
cells from the blood to the surrounding tissues [22, 26]. The leu-
kocyte integrin (lymphocyte function-associated antigen-1, LFA-­
1) binds to ICAM-1 on endothelial cells and directs lymphocytes
to target tissues during inflammation and promotes cell-cell inter-
actions during natural killer (NK) cell- or cytotoxic T lymphocyte
(CTL)-mediated cytotoxicity. Similarly, macrophage-1 antigen
(Mac-1) binding is important in the adherence of monocytes to
endothelial cells and also serves as a complement receptor to
enhance the phagocytosis of opsonized cells or bacteria. Very late
antigens of activation (VLA) are integrins that function during
inflammation to bind immune cells to extracellular matrix (ECM)
proteins such as fibronectin, collagen, and laminin and attract non-
immune cells involved in ECM construction, such as fibroblasts
and endothelial cells [25]. Inflammatory cytokines upregulate the
expression of cell adhesion molecules that serve as ligands for the
integrins ICAM-1, ICAM-2, and vascular cell adhesion molecule-1
(VCAM-1) on endothelial cells in a coordinated fashion, so that
inflammatory cells can be selectively recruited during the various
phases of inflammation. Following firm adhesion to receptors on
 Markers of Inflammation 67

the endothelium, inflammatory cells migrate from the blood vessel

to the tissue by either the paracellular (through an endothelial
junction) or transcellular (directly through the endothelial cell)
route. The exact mechanism that controls transmigration is not yet
defined [22, 26]. Expression of selectins and integrins on cells
changes as the acute phase of an inflammatory response resolves or
progresses into chronic inflammation, and their ligands can be
quantitated using antibody-based labeling techniques such as
immunohistochemistry, flow cytometry, conventional microscopy
techniques, or intravital microscopy. Soluble forms of the selectins
are present in serum and can be measured via ELISA or other
immunoassays. In addition, there are a number of antibody- and
ligand-based functional assays that have been used as research tools
to assess integrin-mediated cell signaling and the efficiency of cell
adhesion [21, 22, 25].

5  Soluble Mediators of the Inflammatory Response

5.1  Cytokines Cytokines act as molecular messengers to coordinate the interplay

between, and control of, different cell types involved in the ampli-
fication and regulation of immune and inflammatory responses. A
single cytokine may affect a number of different cell types or tar-
gets and may have both autocrine and paracrine signaling effects
depending on the target [27]. During innate immune responses,
cytokines are primarily secreted by phagocytic cells and NK cells,
but in adaptive immune responses, they are mainly produced by
antigen-presenting cells (APCs) and lymphocytes. Although often
described separately, cross talk between the innate and adaptive
immune systems is frequent, and cytokines represent a major means
of communication between the various arms of the immune system
[28]. The balance between production of an effective immune
response and tissue damage is dependent on careful regulation of
the cytokine network. The short half-life of cytokines suggests that
under normal conditions most of these soluble mediators are rap-
idly eliminated thereby ensuring their limited bioactivity. However,
during acute and/or chronic inflammatory conditions, cytokines
may be released in sufficient quantity such that systemic effects are
observed [29]. Toxicants that act on either the mediators them-
selves or the cells that produce these mediators may alter the com-
plex interplay of mechanisms that regulate cytokine production,
leading to inflammation and disease. This section provides general
information on many of the cytokines that contribute to inflamma-
tory processes. For a more detailed discussion of the production
and function of the various cytokines, the reader should consult
Chapter 19 (Corsini and House).
Many cytokines and chemokines (see below) contribute
to  inflammation via multiple mechanisms, including facilitating
68 Dori R. Germolec et al.

leukocyte chemotaxis to the site of injury, modulating immune cell

function, and stimulating proliferation and differentiation of the
various cell types involved in the immune response (Table 1). The
cytokines that are best known for stimulating and perpetuating
inflammatory responses are IL-6, IL-1, IL-2, TNF-α, IFN-γ, and
transforming growth factor (TGF)-β [27, 30]. IL-6 is expressed by
a variety of immune cells including, but not limited to, mononu-
clear phagocytes, T cells, B cells, and fibroblasts. Nonimmune cells
may also secrete IL-6; for example, it has been demonstrated that
hepatocytes secrete IL-6 as part of the acute-phase response to
inflammatory stimuli. IL-6 was originally identified as a B-cell dif-
ferentiation factor, as it plays a critical role in the maturation of B
cells into antibody-producing cells, and increased levels of this
cytokine have been associated with polyclonal B-cell activation and
chronic inflammation. IL-6 is also important in the activation and
differentiation of T cells and aids in the regulation of the Th2 and
Treg phenotypes. IL-6 mediates the initial stages of the acute-­
phase inflammatory response, and levels of IL-6 continue to remain
high in chronic inflammatory conditions, leading to enhanced sur-
vival and growth of lymphocytes and macrophages that perpetuate
inflammation [27, 30, 31].
The IL-1 family of cytokines contains 11 different members
that all have both direct and indirect pro-inflammatory effects,
including stimulation of further cytokine production, release of
prostaglandins, generation of cytotoxic effector cells, and synergiz-
ing with colony-stimulating factors to increase the production of
inflammatory cells in the bone marrow. The IL-1 family can be
secreted and expressed by a variety of cell types, including mono-
cytes and macrophages. IL-1β is one of the more prominent pro-­
inflammatory members of the IL-1 family and plays an important
role in the regulation of numerous inflammatory responses. IL-1β
upregulates the gene expression and secretion of inducible nitric
oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2), which
function in the production of downstream inflammatory mediators
(e.g., prostaglandin E2, PAF, nitric oxide). IL-1β also induces the
expression of adhesion molecules which enhance the recruitment
of inflammatory cells [27, 32]. IL-2 augments NK cell activity,
stimulates the production of inflammatory cytokines such as IL-1
and IFN-γ, and enhances macrophage cytotoxicity. It also contrib-
utes to chronic inflammation by stimulating the activation and
proliferation of antigen-specific T and B lymphocytes.
TNF-α is primarily involved in the innate immune response
and enhances inflammation by promoting cytokine production,
inducing the expression of cell adhesion molecules, and stimulat-
ing cell growth and proliferation. TNF-α has been shown to upreg-
ulate the expression of Class I and II major histocompatibility
complex (MHC) molecules on certain cell types, resulting in cell
activation and cytokine release. It is also important in the process
 Markers of Inflammation 69

Table 1
Cytokines and chemokines important in inflammationa

Mediator Source Function

Cytokines
Interleukin 1 (IL-1) Macrophages, dendritic Activates T and B lymphocytes
cells, T and B cells Increases production of other cytokines and
acute-phase proteins
Induces adhesion molecules
Interleukin 2 (IL-2) Activated T cells, B cells Growth and activation of T and B cells
Growth and activation of NK cells and macrophages
Induces production of pro-inflammatory cytokines
Interleukin 6 (IL-6) Macrophages, dendritic Multiple effects on T cells
cells, B cells, activated Myeloid cell development
T cells Regulation of acute-phase proteins
Interleukin 17 TH17 cells, NK cells, Recruits monocytes and neutrophils
(IL-17) NK T cells, macrophages Induces production of many other cytokines,
chemokines, and prostaglandins
Enhances allergic inflammatory responses
Interleukin 33 Macrophages, mast cells, Stimulates production of Th2-associated
(IL-33) dendritic cells, fibroblasts, cytokines from T helper cells, mast cells,
epithelial cells eosinophils, and basophils
Interferon gamma T cells, NK cells, epithelial Increases MHC expression
(IFNγ) cells, fibroblasts Enhances CTL, NK, and macrophage activity
Stimulates production of IL-1 and TNFα
Transforming growth Macrophages, Inhibits cytokine production and activity
factor beta megakaryocytes, Inhibits B-cell proliferation
(TGF-β) chondrocytes Stimulates wound healing
Tumor necrosis Macrophages, dendritic Increases MHC expression
factor alpha cells, lymphocytes, Activates macrophages
(TNFα) mast cells Enhances tumor cell killing
Chemokines
Macrophage Endothelial cells, epithelial Attracts monocytes
chemoattractant cells, fibroblasts, Activates macrophages and T cells
protein-1 (MCP-1) monocytes Stimulates histamine release
(CC family)
RANTES T cells, endothelial cells, Attracts macrophages
(CC family) platelets
Gro (α, β, γ MSGA) Macrophages, fibroblasts Attracts PMS, angiogenesis
(CXC family)
Interleukin 8 (IL-8) Macrophages, lymphocytes Mobilizes PMN from bone marrow
(CXC family)
Fractalkine Endothelial cells, microglia Attracts T cells, monocytes, and PMN in
(CX3C family) macrophages the brain
a
A comprehensive review of cytokines and their functions is beyond the scope of this chapter. The reader is referred to
Chapter 19 of this volume or the recent and thorough review by Akdis et al. [30] for additional information
70 Dori R. Germolec et al.

of removing dead and dying cells through promotion of apoptosis

[27, 30]. IFN-γ is a potent activator of macrophages, stimulates
the production of other pro-inflammatory cytokines such as IL-1
and TNF-α, and also enhances the expression of Class II MHC
molecules on immune cells and vascular endothelium. The latter is
of particular importance, as it is necessary for the transmigration of
inflammatory cells through the vasculature into tissues or to a site
of injury. TGF-β is important in the regulation of tissue repair and
regeneration following injury. It is produced by a number of
immune and nonimmune cell types and aids in the regulation of
the inflammatory response by inhibiting the production of pro-­
inflammatory cytokines such as IL-2, IFN-γ, and TNF-α. TGF-β is
released by platelets following tissue injury and plays an essential
role in wound healing by attracting inflammatory cells, inducing
angiogenesis, and enhancing the deposition of proteins that make
up the extracellular matrix [30].
The relative importance of any individual soluble mediator in a
specific inflammatory condition varies, as cytokines operate in cas-
cades and networks, and target cells may be influenced by multiple
cytokines. While these molecules regulate production of APPs and
other inflammatory mediators, they in turn may be modulated by
transcription factors such as NF-κB and the signal transducers and
activators of transcription (STAT) family [27, 33, 34]. NF-κB,
which has been called a “master transcriptional switch in inflamma-
tion,” regulates dozens of targets involved in inflammation and
other biochemical processes, and changes in NF-κB expression
have also been used as a biomarker of inflammation [35]. Members
of the STAT family, including STAT1, STAT3, and STAT 6, are
involved in the regulation of T-cell differentiation and the activa-
tion of Th1-, Th2- and Th17-driven inflammatory responses [36].

5.2  Chemokines Chemokines are defined based on their amino acid compositiwon,
specifically the presence of a conserved tetra-cysteine motif, and
not by their function as chemotactic cytokines. The relative posi-
tion of the first two consensus cysteines (either separated by a non-­
conserved amino acid or next to each other) provides the basis for
division of chemokines into the two major subclasses, CXC and
CC chemokines, respectively [37, 38]. In general, CC chemokines
serve as chemoattractants for monocytes (RANTES, monocyte
chemoattractant proteins (MCP 1–5)), eosinophils (eotaxins 1–3),
basophils (MCP 4–5), and lymphocytes (macrophage inflamma-
tory protein (MIP)-1α and β). Members of the CXC family, which
includes IL-8, platelet factor 4 (PF4) , and growth-regulated onco-
gene (Gro) α and β, attract PMNs and modulate angiogenesis and
wound healing. Several homologous molecules are also regarded
as chemokines. These are typified by CX3C (fractalkine or neuro-
taxin), which have three intervening amino acids between the first
two cysteines, and XCL1 and XCL2 from the C family of chemo-
kines, which have only a single terminal cysteine.
 Markers of Inflammation 71

Inflammatory chemokines control the recruitment of effector

leukocytes in infection, inflammation, tissue injury, and tumors. As
part of the inflammatory process, chemokines direct cellular migra-
tion, activate macrophages and PMNs, and modulate wound heal-
ing through promotion of angiogenesis and the stimulation of
fibrosis [23]. In addition, chemokines induce the migration of
astrocytes to, and microglial cell proliferation at, sites of injury in
the central nervous system, which promotes the transmission of
nociceptive pain signals that serve as a warning sign following acute
tissue damage [39]. Cell migration, or chemotaxis, often occurs
along chemokine gradients, and changes in chemokine receptor
levels on leukocytes and endothelial cells regulate localization of
leukocytes to sites of inflammation [37]. Chemokines share recep-
tors with other chemotactic molecules such as PAF, C5a (see
below), and leukotriene B4. Activated effector or memory T cells
are the source of multiple inflammatory chemokines, and by sus-
taining effector T-cell and macrophage recruitment, these inflam-
matory chemokines can subsequently control the local production
of pro-inflammatory cytokines [40]. Thus, locally produced
­chemokines can influence disease progression and pathogenesis.
Increased levels of chemokines have been associated with both
acute and chronic inflammatory conditions, and a number of
chronic inflammatory diseases including atherosclerosis and glo-
merulonephritis. Numerous tools are available for evaluating the
role and function of chemokines and their receptors in inflamma-
tion and disease. As indicated above, one of the more common
methods for evaluating localized mediators in a composition of
inflammatory infiltrates is immunohistochemical (IHC) staining of
relevant tissue sections. There are a number of monoclonal anti-
bodies available for the characterization and measurement of che-
mokines and their receptors. In addition, chemokine function can
be measured in cell-based assays that assess the migration of cells
across filters or membranes in response to chemoattractants [41].

5.3  Acute-Phase Inflammation typically triggers an acute-phase response, which

Proteins results in changes in blood proteins known as acute-phase proteins
(APPs). Production of APPs occurs primarily in the liver, and
increased (positive APPs) or decreased (negative APPs) production
and release occur in response to cytokine signals from the site of
inflammation. These proteins alter homeostasis in order to initiate
or support defensive and/or adaptive processes that contribute to
healing in the short term, but can lead to chronic inflammation,
metabolic disturbances, and tissue damage with prolonged stimula-
tion. Positive APPs include C-reactive protein (CRP), amyloids A
and P, ceruloplasmin, haptoglobin, alpha 1-acid glycoprotein, alpha
2-macroglobulin, complement components and coagulation pro-
teins such as Factors V and VIII, and fibrinogen. Negative APPs
include albumin and alpha-1 apolipoprotein [42]. Because these
proteins are regulated in response to inflammatory signals and
72 Dori R. Germolec et al.

change rapidly as conditions change, they are good markers of

inflammation. It should be noted that APPs may increase as part of
the stress response [15], so APP data should be interpreted in the
context of other available information. Traditionally, APPs are mea-
sured directly in plasma or serum and are used clinically to evaluate
the presence, intensity, and course of an inflammatory process or
disease. For individual APPs, basal levels and the degree of partici-
pation in the acute-phase response vary across species, so the most
relevant marker for one species may not have relevance in another
[42–45]. In addition, some available assays are species-­specific, and
reagents may not be available for a given APP in the species of inter-
est. To increase sensitivity, it is best to determine the most appropri-
ate APP for the species in question, or to assess a panel of APPs.
The most sensitive and commonly recognized diagnostic tool
for the acute-phase response in humans is CRP; however, CRP is of
variable importance in species commonly used for toxicology stud-
ies [43]. CRP is a major responder and useful biomarker of acute-
phase response in dogs, nonhuman primates, and pigs [44, 45].
Hepatic CRP synthesis is regulated by pro-inflammatory cytokines
including IL-6, IL-1, and TNF-α; therefore, almost any nonspecific
tissue injury, infection, inflammation, or stress can be associated
with increased circulating CRP values [46]. CRP shares several
functions with Ig molecules, in that it can opsonize microorganisms
such as bacteria and fungi through binding to cell wall polysaccha-
rides, as well as facilitate phagocytosis through its ability to activate
and fix complement. Conventional CRP immunoassays have been
shown to be more reliable indicators of acute inflammation than
leukocyte counts, as blood levels of CRP rise and fall rapidly in a
reflection of the disease process [47]. Although somewhat contro-
versial, CRP has been shown to be a good predictor of cardiovascu-
lar disease risk, hypertension, and diabetes in humans [48–51].
Serum amyloid A is a major participant in the inflammatory
process in dogs, mice, and pigs. Additional APPs which are useful
for studies in pigs are porcine major APP and haptoglobin. In
mice, in addition to serum amyloid A, the major APPs are hapto-
globin and serum amyloid P.  For rats, a panel including α1-acid
glycoprotein and α2-macroglobulin would be more appropriate
than CRP [44].
Fibrinogen is an APP that has significant cross-species relevance
and is easily incorporated into standard preclinical toxicity studies.
It must be measured in plasma, not serum, as it is consumed in the
formation of the blood clot. The coagulation process is similar to
that of the complement system (see below) in that it operates as a
series of transformations of proenzymes to activated enzymes. This
process culminates in the formation of thrombin, which converts
soluble fibrinogen into insoluble fibrin. Thus, fibrinogen provides
an interface between inflammation and coagulation, as higher levels
of fibrinogen are reflective of inflammation [52]. These enzymatic
 Markers of Inflammation 73

reactions normally occur on the surface of activated endothelial

cells and platelets. Cross talk between proteins in the complement
and clotting cascades occurs at a number of points, either through
interactions with target cells or through processes involving inflam-
matory mediators other than complement effectors [53]. Receptors
for fibrinogen have been identified on monocytes/macrophages,
neutrophils, platelets, and NK cells, and the binding of fibrinogen
activates signaling pathways that upregulate NF-κB expression and
the release of pro-inflammatory cytokines [54]. Peptides formed
during the cleavage of fibrinogen increase vasodilation and serve as
chemoattractants, allowing fibrinogen to act as a bridging molecule
between leukocytes and endothelial cells facilitating the recruitment
of these cells to the site of inflammation. Along with CRP, serum
levels of fibrinogen have been used as a predictor of cardiovascular
disease risk, hypertension, and diabetes in humans [51, 55]. It
should be noted that fibrinogen is utilized at an accelerated rate in
situations of DIC, which may occur during severe inflammatory
processes. In the face of DIC, plasma fibrinogen decreases and
forms fibrin degradation products (FDPs), which may mask the
increase seen in inflammation and vice versa. The measurement of
FDPs or a subset of FDPs known as D-dimers, along with assess-
ment of other parameters suggesting inflammation (leukogram,
other APPs) or DIC (thrombocytopenia, prolonged clotting times),
is useful in interpreting fibrinogen data.

5.4  The Complement The complement system is a complex network of proteins that par-
System ticipate in the acute inflammatory response through their enzy-
matic activity, effects on mediator release, chemotaxis and vascular
permeability, and the ability to enhance phagocytosis through
opsonization of microbes. A highly simplified summary of the
complement system is presented in Fig. 2, and the reader is referred
to basic texts such as Janeway’s Immunobiology [56] for a more
comprehensive discussion.
The classical arm of the complement system is activated by
antigen-antibody interactions, which trigger a cascade of reactions,
each of which results in the activation of another complement
component. There are two antibody-independent pathways that
can trigger complement activation. Several lectins, including
mannose-­binding lectin and ficolin, are structurally similar to the
C1 proteins that initiate the classical complement cascade [57].
These lectins can activate complement by cleaving the C2 and C4
proteins, in a manner similar to that of C1, leading to the cleavage
of C3, a common step in all three pathways of the complement
network.
Microbial cell wall proteins such as LPS and zymosan (from
yeast) activate the alternate complement pathway by interacting
with three triggering factors (initiating factor, factor B and factor
D) that combine to cleave C3. C3 is the most abundant of the
74 Dori R. Germolec et al.

Fig. 2 Overview of complement activation. This figure summarizes the three major pathways of complement
activation and formation of the membrane attack complex. Abbreviations: MBL mannose-binding lectin, MASP
MBL-associated serine protease

complement proteins and plays a central role in the complement

pathway [58]. Cleavage of C3 releases a small peptide, C3a, which,
along with C5a, increases vascular permeability allowing the influx
of inflammatory cells and proteins. The larger cleavage product,
C3b, binds to the surface of pathogens, as well as to complement
receptors on inflammatory cells, enhancing cell adhesion and
phagocytosis. C3b also binds to the activated products from earlier
steps in the classical/lectin (C4b2a) or alternate (Bb) pathways to
form the C5 convertase which cleaves the C5 protein into C5a, the
most potent of the peptide mediators of local inflammation, and
C5b, which triggers the terminal events in the complement cascade
resulting in the formation of the cytolytic membrane attack com-
plex (MAC).
The formation of immune complexes in autoimmune diseases
activates complement resulting in chronic inflammation and tissue
damage. Deposition of complement is frequently used as an immu-
nohistochemical marker of inflammation, and the levels of indi-
vidual complement factors can be quantitated in serum. The
activity of serum complement can be determined in the laboratory
by evaluating its ability to lyse cells in  vitro. Assessment of the
activity of individual complement components is not routinely
 Markers of Inflammation 75

used as a marker of inflammation, but is more commonly used as a

diagnostic tool for complement deficiencies or to identify direct or
indirect activation of complement by chemicals, pharmaceuticals,
drugs/antidrug antibody complexes, or novel therapeutic modali-
ties such as nanoparticles.

6  Molecular Methods for Evaluating the Inflammatory Response

Recently “omics” technologies have been used to detect APPs asso-

ciated with inflammation in human and animal studies. Consistent
with conventional measures of APPs, serum and plasma are com-
monly used matrices for detection of circulating proteins by two-
dimensional electrophoresis (2DE) and mass spectrometry (MS).
These techniques can provide a more complete picture of the inflam-
matory process, since they can separate and measure active proteins,
precursors, metabolites, and degradation products [59, 60].
Upregulation of a number of APPs, including CRP [59, 61], α-2-
macroglobulin [62], complement C3 [62], serum amyloid A [63],
and plasminogen [59], has been identified through 2DE and/or
MS in association with inflammatory conditions in both clinical and
laboratory animal studies. Ironically, several of the most abundant
APPs, including fibrinogen, haptoglobin, and α-1-antitrypsin, are
often removed prior to proteomic analysis because their strong sig-
nals mask that of other proteins [59, 64, 65].
Genomic techniques, especially polymerase chain reaction
(PCR) and microarray, have been used to measure the expression
of cytokines, chemokines, adhesion molecules, and ligands. In
addition to evaluating individual molecules, pathway analysis pro-
vides vital information on the mechanism and signaling events of
inflammation and inflammatory disease. For example, the STING
(stimulator of interferon (IFN) genes) pathway stimulates the tran-
scription of IFN and other innate immune genes in response to
viruses, bacteria, DNA, and other microbial products in endothe-
lial, epithelial, and hematopoietic cell types [66]. The copy number
and mutation of mitochondrial DNA (mt DNA) is currently being
investigated to determine the potential correlation with inflamma-
tion and disease in both human patients and in mouse models.
Although changes in hematology dynamics, APPs, complement
factors, and cytokines are common to virtually all inflammatory
conditions, reliable, individual biomarkers associated with specific
pathologic events are available for only a limited number of circum-
stances. High-content technologies, such as gene and protein
microarrays, are necessary for gene/protein screening and model
development. DNA microarrays have been used to conduct a multi-
organ, multi-pathway study examining the effects of hexachloro-
benzene (HCB) in rats [67]. These studies identified increased
transcript levels of markers for granulocytes and macrophages that
76 Dori R. Germolec et al.

were supported by histopathological findings. Upregulation of

genes encoding pro-inflammatory cytokines and chemokines, APPs,
and complement components was also observed, suggesting that
HCB induces a strong, systemic i­nflammatory response. In addition
to the immune response genes, this study also identified altered
functions and pathways associated with metabolism, oxidative
stress, and reproductive toxicity, and it enabled the relative com-
parison of effects in the spleen, liver, kidney, blood, mesenteric
lymph nodes, and thymus.
Since most APPs and complement factors are produced in the
liver, rather than directly in the affected tissue, they do not help to
identify the specific target tissue(s) or provide an accurate reflec-
tion of the extent of damage. However, upregulation of transcrip-
tion/translation of inflammatory mediators in the liver may be an
informative component in short-term toxicity screens that could
trigger further investigation into the specific nature of the inflam-
matory response. If damage to target tissue is easily assessed by
clinical pathology data (e.g., hepatocellular leakage of enzymes
such as ALT and AST in the case of hepatitis), the damaged tissue
may be identified; however, generally blood chemistry is unre-
warding in identifying sites of inflammation.
Protein arrays and a support vector machine algorithm have
been used to identify an inflammatory signature expression pattern,
consisting primarily of cytokines and chemokines, that could suc-
cessfully predict the stage of atherosclerosis in an independent data
set [68]. The investigators evaluated proteins found in serum and
validated the potential markers using quantitative real-time reverse
transcriptase polymerase chain reaction (RT-PCR) of RNA from the
aortic vascular wall to discover a profile specific to atherosclerosis.

7  Conclusions

Proteomic and genomic technologies allow simultaneous analysis

of the complement factors, cytokines and chemokines, apolipopro-
teins, and other APPs, as well as tissue-specific proteins and pro-
teins associated with other physiological functions to provide a
more integrated picture of inflammation, mechanisms of action,
and the disease process [65]. Transcriptomic profiles offer the pos-
sibility of unifying traditional diagnostic markers of inflammation
with regulatory and immune-related molecules to provide tempo-
ral, mechanistic, and predictive information. The protein and gene
profiles of inflammation vary with each disease or condition, and
several studies indicate that multiple molecules representing differ-
ent biochemical pathways and pathophysiologic processes offer the
best strategy for identifying gene signatures representative of
inflammatory changes [68–70].
 Markers of Inflammation 77

Acknowledgments

The authors would like to thank Dr. Georgia Roberts and Dr.
Gregory Kane for their thoughtful and comprehensive review of
this chapter. This work was supported in part by the Intramural
Research Program of the National Institute of Environmental
Health Sciences, National Institutes of Health.

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