Objective—To evaluate the activation status of neutrophils
in blood samples obtained from horses with
naturally occurring colic associated with strangulating
obstruction, nonstrangulating obstruction, or inflammatory
Animals—30 horses with naturally occurring colic
and 30 healthy control horses.
Procedure—Activation status of neutrophils was
determined by assessing the number of neutrophils
that could pass through filters with 5-µm pores, cellsurface
CD11-CD18 expression, and alterations in size
and granularity of neutrophils.
Results—Horses with impaction or gas colic did not
have evidence of activated neutrophils. Horses with
inflammatory bowel disease consistently had evidence
of activated neutrophils, including decreased
leukocyte deformability, increased CD11-CD18 expression,
increased neutrophil size, and decreased neutrophil
granularity. Horses with strangulating colic had
variable results. Of horses with strangulating colic, 7
of 14 had marked changes in filtration pressures, 5 of
14 had increased CD11-CD18 expression, 6 of 14 had
changes in neutrophil size, and 5 of 14 had changes in
neutrophil granularity. Among horses with strangulating
colic, changes in deformability, size, and granularity
of neutrophils correlated with an adverse outcome.
Conclusions and Clinical Relevance—Activated
neutrophils were detected in all horses with inflammatory
bowel disease and a few horses with strangulating
colic. Correlation of activated neutrophils
with horses that had strangulating colic that died or
were euthanatized indicates that activated neutrophils
are a negative prognostic indicator. Additional
studies are needed to determine whether activated
neutrophils contribute directly to the adverse outcome
in horses with strangulating colic. (Am J Vet Res 2003;64:1364–1368)
Objective—To evaluate lipopolysaccharide (LPS)-
induced activation of equine neutrophils in blood.
Sample Population—Blood samples from 5 healthy
Procedure—Neutrophil integrin (CD11/CD18)
expression, size variation, degranulation, and
deformability were measured with and without incubation
with LPS. Time and concentration studies were
done. The mechanism of endotoxin-induced neutrophil
activation was investigated by inactivating
complement or preincubating neutrophils with
inhibitors of tumor necrosis factor-α (TNF-α) synthesis,
prostaglandin-leukotriene synthesis, or plateletactivating
Results—Incubation of equine neutrophils with LPS
increased cell surface expression of CD11/CD18,
decreased neutrophil deformability, increased and
decreased neutrophil size, and induced neutrophil
degranulation. The LPS-induced neutrophil activation
was attenuated by addition of inhibitors of TNF-α and
Conclusion and Clinical Relevance—Equine neutrophils
are readily activated in vitro by LPS, resulting
in increased expression of integrin adhesion molecules,
decreased deformability, variation in neutrophil
size, and degranulation. The tests used to detect activated
neutrophils in this study may be useful in
detecting in vivo neutrophil activation in horses with
sepsis and endotoxemia. (Am J Vet Res
Objective—To determine functional characteristics of
monocytes obtained from cows with subclinical infection
with Mycobacterium avium subsp paratuberculosis
(MAP) that may have predisposed those cows to
becoming infected with MAP.
Sample Population—Monocytes obtained from 5
uninfected cows and 5 cows subclinically infected
with MAP in a herd with a high prevalence of paratuberculosis
(ie, Johne's disease).
Procedures—Monocytes from uninfected and subclinically
infected cows were incubated with MAP for
2, 6, 24, 72, or 96 hours. Variables measured included
expression of tumor necrosis factor-α (TNF-α), interleukin
(IL)-10, IL-12, transforming growth factor-β, and
suppressor of cytokine signaling-3 (SOCS-3); apoptosis
of monocytes; acidification of phagosomes; and
killing of MAP.
Results—Monocytes from infected cows had greater
expression of IL-10 and SOCS-3 at 2 hours of coincubation
with MAP and lower expression of TNF-α and
IL-12 when results for all incubation times were combined.
Monocytes from infected cows had a greater
capacity to acidify phagosomes. No differences were
observed in the rate of apoptosis or capacity of monocytes
to kill MAP organisms.
Conclusions and Clinical Relevance—Monocytes
obtained from cows with subclinical infection with
MAP had upregulated expression of IL-10 and
SOCS-3 within the first 2 hours after exposure to MAP
organisms. Although this did not inhibit acidification of
phagosomes, apoptosis of monocytes, or attenuation
of the capacity to kill MAP organisms, it may have
attenuated the capacity of mononuclear phagocytes to
initiate inflammatory and adaptive immune responses.
(Am J Vet Res 2005;66:1114–1120)
Objective—To evaluate activation of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in bovine monocytes after incubation with Mycobacterium avium subsp paratuberculosis (Mptb) organisms.
Procedures—Bovine monocytes were incubated with Mptb organisms with or without a specific inhibitor of the JNK/SAPK pathway (SP600125) for 2, 6, 24, or 72 hours. Expression of interleukin (IL)-1β, IL-10, IL-12, IL-18; transforming growth factor-β (TGF-β); and tumor necrosis factor-α (TNF-α) and the capacity of Mptb-infected monocytes to acidify phagosomes and kill Mptb organisms were evaluated. Phosphorylation status of JNK/SAPK was evaluated at 10, 30, and 60 minutes after Mptb incubation.
Results—Compared with uninfected control monocytes, Mptb-infected monocytes had increased expression of IL-10 at 2 and 6 hours after incubation and had increased expression of TNF-α, IL-1β, IL-18, and TGF-β at 2, 4, and 6 hours. Additionally, Mptb-infected monocytes had increased expression of IL-12 at 6 and 24 hours. Addition of SP600125 (specific chemical inhibitor of JNK/SAPK) resulted in a decrease in TNF-α expression at 2, 6, and 24 hours, compared with untreated Mptb-infected cells. Addition of SP600125 resulted in a decrease in TGF-β expression at 24 hours and an increase in IL-18 expression at 6 hours. Addition of SP600125 failed to alter phagosome acidification but did enhance the capacity of monocytes to kill Mptb organisms.
Conclusions and Clinical Relevance—Activation of JNK/SAPK may be an important mechanism used by Mptb to regulate cytokine expression in bovine monocytes for survival and to alter inflammatory and immune responses.
Objective—To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPKERK) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes.
Sample Population—Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms.
Procedures—Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPKERK pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-α and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated.
Results—The MAPKERK pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-α and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes.
Conclusions and Clinical Relevance—Results indicated that the MAPKERK pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-α and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPKERK pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle.
Objective—To develop an in vitro model of the bovine
alveolar-capillary interface and to evaluate the roles of
interleukin-8 (IL-8) and platelet-activating factor (PAF)
in neutrophil-mediated endothelial injury induced by
infection with Mannheimia haemolytica.
Procedure—A coculture system was developed in
which endothelial cells were grown to confluence in
tissue culture inserts, neutrophils were added to the
inserts, and macrophages were added to tissue culture
wells. Mannheimia haemolytica-derived
lipopolysaccharide (LPS) or supernatant was added to
activate macrophages, and inhibitors of PAF or IL-8
were added to the insert. Endothelial cell cytotoxicity
and permeability (ie, albumin leakage) and neutrophil
activation (ie, adhesion, degranulation [lactoferrin
expression], and superoxide production) were
Results—The addition of M haemolytica-derived LPS
to bovine macrophages in the coculture system
resulted in significant increases in endothelial cell
cytotoxicity and permeability and neutrophil degranulation
and adhesion. Inhibition of IL-8 reduced
endothelial cell permeability and neutrophil degranulation
induced by exposure to M haemolytica-derived
supernatant, whereas inhibition of PAF decreased
superoxide release by neutrophils.
Conclusions and Clinical Relevance—In vitro activation
of bovine macrophages by M haemolyticaderived
LPS resulted in neutrophil activation and neutrophil-
mediated endothelial damage. Neutrophilmediated
endothelial injury and neutrophil degranulation
were, at least in part, mediated by IL-8, whereas
PAF promoted superoxide release by neutrophils in
this in vitro system designed to mimic the in vivo
events that occur during the early stages of bovine
pneumonic pasteurellosis. (Am J Vet Res 2002;
Objective—To evaluate the role of the nuclear factor-κB (NF-κB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP).
Sample Population—Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection.
Procedures—Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-κB pathway (pyrrolidine dithiocarbamate), and activation of the NF-κB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated.
Results—Addition of MAP organisms to monocytes activated the NF-κB pathway as indicated by increased NF-κB–DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-κB, decreased expression of TNF-α and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells.
Conclusions and Clinical Relevance—Results indicated that NF-κB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-κB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.
Objectives—To evaluate the role of interleukin (IL)-10
in the inability of monocyte-derived bovine
macrophages to kill Mycobacterium avium subsp
paratuberculosis organisms in vitro.
Sample Population—Monocytes were obtained
from healthy adult Holstein dairy cows that had negative
results when tested for infection with M avium
Procedure—Monocyte-derived macrophages were
incubated with M avium subsp paratuberculosisfor 2,
6, 24, 72, or 96 hours with or without addition of saturating
concentrations of a goat anti-human IL-10 that
has been documented to neutralize bovine IL-10 activity.
Variables assessed included ingestion and killing
of M avium subsp paratuberculosis; expression of
tumor necrosis factor (TNF)-α, IL-12, IL-8, major histocompatability
(MHC) class II, vacuolar H+ ATPase,
and B cell CLL/lymphoma 2 (BCL-2); production of
nitric oxide; acidification of phagosomes; and apoptosis
Results—Neutralization of IL-10 enabled macrophages
to kill 57% of M avium subsp paratuberculosis organisms
within 96 hours. It also resulted in an increase in
expression of TNF-α, IL-12, IL-8, MHC class II, and vacuolar
H+ ATPase; decrease in expression of BCL-2;
increase in acidification of phagosomes; apoptosis of
macrophages; and production of nitric oxide.
Conclusions and Clinical Relevance—The capacity
of M avium subsp paratuberculosis to induce IL-10
expression may be a major determinant of virulence
for this organism. (Am J Vet Res 2005;66:721–726)
Objective—To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms.
Sample Population—Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results.
Procedures—Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry.
Results—Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect.
Conclusions and Clinical Relevance—Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.
Objective—To determine whether platelet-activating
factor (PAF) is involved in acute lung microvascular
injury associated with pneumonic pasteurellosis in
Animals—15 healthy 2- to 4-week-old male Holstein
Procedure—Calves were anesthetized and inoculated
intrabronchially with saline (0.9% NaCl) solution
(n = 5) or 1 × 109Pasteurella haemolytica organisms
(n = 10). Of the 10 calves inoculated with P haemolytica,
5 also were treated with WEB 2086, a potent
inhibitor of PAF, and 5 were treated with vehicle.
Blood and bronchoalveolar lavage samples were collected
before and 1, 2, 4, and 6 hours after inoculation
of P haemolytica. Blood samples were analyzed to
evaluate total number and differential counts of leukocytes,
dilute whole-blood leukocyte deformability,
size of neutrophils, and neutrophil CD11b expression.
Bronchoalveolar lavage samples were analyzed for
total number and differential counts of nucleated
cells, total protein concentration, and hemoglobin
concentration. Size and gross and histologic appearance
of lung lesions also was determined.
Results—Treatment of calves with WEB 2086
reduced size of lung lesions, attenuated the increase
in microvascular permeability, and reduced neutrophil
infiltration in the first 4 hours after inoculation.
Treatment with WEB 2086 also attenuated a
decrease in leukocyte deformability, increase in size
of neutrophils, and CD11b expression by circulating
Conclusions and Clinical Relevance—It appears that
PAF is a major mediator for altered lung microvascular
permeability and activation of circulating neutrophils in
the first 4 hours after onset of pneumonic pasteurellosis
in calves. (Am J Vet Res 2000;61: