Previous Article | Next Article 
Infection and Immunity, December 1999, p. 6293-6302, Vol. 67, No. 12
Department of Veterinary Molecular Biology,
Montana State University, Bozeman, Montana 59717
Received 16 June 1999/Returned for modification 20 July
1999/Accepted 15 September 1999
During bacterial infection of the bovine mammary gland, large
numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the
specific leukocyte populations mediating this immune response are not
well defined. In the studies described here, we analyzed blood and milk
from healthy cows and cows with naturally occurring mastitis to
determine if distinct Despite increased educational
efforts and improved dairy herd management, mastitis still represents
one of the most costly diseases of the dairy industry (53).
In fact, the yearly loss due to mastitis has recently been estimated at
about $2 billion for dairy producers in the United States alone
(15, 25). In the common subclinical or chronic cases,
mastitis can persist for months with little obvious inflammation.
However, many of these infections eventually develop into clinical
mastitis, which results in acute or slowly progressing inflammation and
can later end in fibrosis of mammary tissue and loss of or decrease in
milk production (53).
The most common bacterial pathogens associated with mastitis include
staphylococcal, streptococcal, and coliform bacteria (15,
25). Staphylococcus aureus is currently one of the
most difficult pathogens to control because it can spread rapidly among the herd and responds poorly to conventional antibiotic therapy (37). Members of another common group of mastitis-causing
bacteria, Streptococcus spp., are frequently present on
mucous membranes and are extremely infectious for the bovine mammary
gland. Streptococcal mastitis causes a persistent type of infection
that does not have a high self-cure rate, and undetected or untreated
infected cattle can serve as reservoirs of infection (25,
60).
In efforts to prevent mastitis, a number of vaccines which can reduce
the severity of mastitis have been generated; however, these vaccines
still fail to effectively prevent the development of mastitis
(67). Thus, the identification of alternative methods for
combating mastitis is essential. In this regard, one of the most
practical means for dealing with mastitis in the dairy industry may be
to enhance the natural host defense mechanisms of the animal (29). Strategies aimed at enhancing the immune responses of the mammary gland during infection would significantly affect the
ability of the animal to resist infection. Currently, the roles of
various immune system components in the defense of the mammary gland
against infection are not well understood. Both cytokine production and
leukocyte adhesion play important roles during bacterial infection
(29); however, the relative contributions of these factors
to the pathogenesis of mastitis are not yet fully determined and will
require more extensive studies. In addition, the contributions of
various lymphoid and myeloid subsets to host defense in the mammary
gland have not been extensively evaluated with naturally infected cows.
Park et al. (41) reported that the presence of increased
T-lymphocyte levels in bovine milk during lactation was due to an
increase in the number of activated CD8+ T cells. In
subsequent studies, Park et al. (42) showed that the number
of activated CD8+ T cells was increased in milk obtained
from cows experimentally infected with S. aureus and that
these cells were responsible for suppressing the proliferative response
of milk CD4+ T cells. Taylor et al. (56) also
found that T cells in bovine milk were predominantly CD8+;
however, as the number of days of lactation increased, the number of
CD4+ T cells increased. In addition, Taylor et al.
(57) also reported an increased percentage of
CD4+ T cells in milk from cows with mastitis. Together,
these previous reports demonstrate that changes in milk lymphocyte
populations occur during mastitis; however, the nature of these changes
seems to vary depending on the pathogen. In addition, because
antibodies recognizing distinct In the studies described here, we have investigated the hypothesis that
distinct Animals.
Thirty Holstein dairy cows from a local dairy were
used throughout these studies. Control blood and milk samples were
taken from apparently healthy animals. Acute mastitis was identified by
detectable signs of inflammation of the infected udder and visual
changes in infected milk. Clinical findings were confirmed by somatic
cell counts in the foremilk and by bacteriologic culturing of milk from
suspect quarters as described below. Animal care and handling were
carried out in accordance with institutional guidelines.
Leukocyte isolation from blood.
Bovine lymphocytes and
neutrophils were isolated from bovine blood as described by Sipes et
al. (51). Briefly, blood was obtained by jugular venous
puncture and collected in 20-ml Vacutainer tubes containing 0.2 ml of
0.5 M EDTA. After removal of erythrocytes by H2O lysis,
leukocytes were resuspended in 20 ml of cold Dulbecco's phosphate-buffered saline (DPBS) and layered onto a two-step Histopaque gradient consisting of 15 ml of Histopaque 1077 over 15 ml of a 1:1
mixture of Histopaque 1077 and Histopaque 1119. After centrifugation at
2,500 × g for 25 min at room temperature, the two
layers of cells were collected separately. The purified cells were
washed two times with 20 ml of cold DBPS. Based on Wright staining and microscopic analysis, cells were routinely determined to be >95% pure; viability was determined to be >98% based on the exclusion of
trypan blue. The final cell concentration used for flow cytometric analysis was 107 cells/ml.
Leukocyte isolation from milk.
Lymphocytes and neutrophils
were isolated separately from the milk of 30 lactating cows. Briefly,
milk was aseptically collected into sterile flasks and kept on ice
until used. The milk was centrifuged at 1,500 × g for
20 min at 4°C, the cream layer was removed with a spatula, and the
milk was gently decanted. The cell pellet was resuspended in 20 ml of
ice-cold DPBS and filtered through 30-µm-pore-size Nitex to remove
any remaining debris. The milk cell suspension was then applied to a
Histopaque gradient as described above. Leukocytes or purified
lymphocyte and neutrophil fractions were washed twice with DPBS and
used for flow cytometric analysis.
Flow cytometry.
Single-color flow cytometric analysis was
performed as described previously (21). Briefly,
106 cells were incubated with 50 µg of primary antibody
per ml for 30 min on ice, washed with phosphate-buffered saline-2%
goat serum, and incubated for 30 min with 1:250-diluted fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G
secondary antibody (Jackson ImmunoResearch, West Grove, Pa.). The cells
were washed again, resuspended in phosphate-buffered saline-2% goat
serum, and analyzed by flow cytometry with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). A total
of 10,000 events were collected for each sample. All data files were
further analyzed with CellQuest software (Becton Dickinson).
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Selective Recruitment of T-Cell Subsets to the
Udder during Staphylococcal and Streptococcal Mastitis: Analysis of
Lymphocyte Subsets and Adhesion Molecule Expression
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and 
T-lymphocyte subsets were
involved in the response of the udder to a mastitis pathogen and if the
type of mastitis pathogen influenced the subset composition of these
responding leukocytes. Although blood samples from cows with confirmed
staphylococcal and streptococcal mastitis were characterized by
increased numbers of T cells, the most dramatic changes in
leukocyte distributions occurred in milk samples from these cows, with
a 75% increase in T-cell levels and a 100% increase in
T-cell levels relative to the levels in milk samples from healthy
animals. Interestingly, the increase in T-cell numbers observed
in milk from cows with staphylococcal mastitis was primarily due to
increased numbers of CD4+ T cells, while the increase in
T-cell numbers observed in cows with streptococcal mastitis was
due to a parallel increase in both CD4+ and
CD8+ T-cell numbers. The increased numbers of T
cells in milk from cows with staphylococcal and streptococcal mastitis
were due to a selective recruitment of a distinct T-cell subset
(GD3.1+), while no change in the numbers of
GD197+ T cells was observed. We also analyzed
adhesion protein expression on blood and milk leukocytes and found
that, in comparison to the situation for healthy cows, L-selectin was
down-regulated and CD18 was up-regulated on leukocytes from cows with
mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by
neutrophils may provide a sensitive indicator of early inflammatory
responses during bovine mastitis. Overall, these studies suggest that
distinct and T-cell subsets are involved in the host
defense of the udder against mastitis infection and that selective
recruitment of these T-cell subsets depends on the infectious agent involved.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
T-lymphocyte subsets have only
recently been developed (66), the participation of these
T-lymphocyte subsets in mastitis has not been evaluated.
Clearly, a more comprehensive understanding of these factors of the
bovine immune response is essential to the development of effective
treatments for the prevention of mastitis.
and T-lymphocyte subsets are involved in the
response of the udder to a mastitis pathogen and that the type of
pathogen may influence the subset composition of these responding
leukocytes. To evaluate this hypothesis, we have used a panel of
monoclonal antibodies to characterize the lymphocyte populations
present in normal and mastitis milk with respect to leukocyte subset
distribution and adhesion molecule expression. In addition, we have
provided a comparison of these parameters for cows naturally infected
with streptococcal or staphyloccocal bacteria. These studies have
helped to delineate the role of the various leukocyte subsets in the
host defense of the bovine mammary gland against infection with these
different pathogens.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
T-cell receptors (TCRs) (GD3.8) (66), T-cell receptor subsets (GD3.1 and GD197) (18, 66),
CD2 (CC42) (38), CD4 (CC30) (38), CD8 (CC58)
(38), bovine neutrophils (BN15.6) (52),
L-selectin (Dreg-56) (63), and CD18 (MHM23) (34).
Although T cells have been shown previously to express CD2
(31, 65, 66), this staining represents only a minor subset
of T cells (~5%) (65). Additionally, we confirmed that the level of CD2+ T cells was negligible in our
samples (e.g., see Fig. 3). Therefore, we used CD2 staining to define
and quantify the T-cell population in these studies. Neutrophils
were identified by their distinct forward and side light scatter
profiles and by positive flow cytometric staining with antibody BN15.6,
an antibody specific for bovine neutrophils (52).
Somatic cell counting. Somatic cell counts were determined by the State of Montana Veterinary Diagnostic Milk Laboratory (Bozeman). Cell counts were determined for all milk samples by use of a FOSSOMATIC electronic cell counter (Foss America Inc., Fishkill, N.Y.).
Bacteriology. Bacteriologic analysis was performed on all milk samples by the State of Montana Veterinary Diagnostic Bacteriology Laboratory. Briefly, 0.01 ml of each milk sample was placed on tryptose agar containing 5% bovine blood. Plates were incubated for 48 h at 37°C, and the presence of pathogenic bacteria was considered a positive indicator of infection.
Statistical analysis. Statistical analysis was performed by a paired Student t test, and a P value of <0.05 was considered significant.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Leukocyte distribution in blood and milk of healthy and mastitic
cows.
In blood from both healthy and infected animals, the overall
total leukocyte counts were not significantly different, irrespective of the mastitis pathogen (Table 1).
Furthermore, the leukocyte subset distribution in blood obtained from
healthy cows was similar to that in blood obtained from cows with
confirmed staphylococcal or streptococcal mastitis, with one important
exception: blood from cows with mastitis had significantly increased
numbers of T cells (Fig. 1).
|
|
and T-cell numbers as well (Fig. 1). In addition,
the relative T-cell/ T-cell ratios increased from an
average of 0.37 in healthy cows to 0.42 and 0.44 in cows with
staphylococcal mastitis and streptococcal mastitis, respectively. These
results suggested the interesting possibility that selective
recruitment of T-cell subsets to the udder might occur during mastitis.
Thus, we performed further studies to investigate the relative
and T-cell subset distributions in milk from these animals.
Lymphocyte subset distribution in milk of healthy and mastitic
cows.
As shown in Fig. 1, milk samples from cows with
staphylococcal and streptococcal mastitis contained approximately 75%
more T cells than and twice as many T cells as samples
from healthy cows. Further analysis of the T-cell subsets responsible
for these changes showed that different and subsets were
selectively recruited, depending on the mastitis pathogen (Fig.
2). In milk from cows with staphylococcal
mastitis, the increase in T-cell numbers was due primarily to a
selective increase in CD4+ T-cell numbers, changing the
CD4+/CD8+ ratio from 0.68 in healthy cows to
1.39 in mastitic cows (Fig. 2). In contrast, the increase in
T-cell numbers in milk from cows with streptococcal mastitis, which was
similar in amplitude to that in cows with staphylococcal mastitis, was
due to increases in both CD4+ and CD8+ T-cell
numbers, without a major change in either subset relative to the other
(CD4+/CD8+ ratio, 0.65).
|
T-cell subsets in milk samples from cows with
streptococcal and staphylococcal mastitis showed that the increase in
T-cell numbers shown in Fig. 1 was due, in part, to
significantly increased levels of GD3.1+ T cells,
while the levels of GD197+ T cells remained similar
to those in milk samples from healthy cows (Fig. 2). The remaining
increase in T-cell numbers appeared to be due to a variable
increase in CD8+ T-cell numbers in the milk of
mastitic cows (5 to 20%). This observation is consistent with the
studies of Park et al. (43), who reported the presence of
elevated numbers of CD8+ T cells in the milk of cows
with staphylococcal mastitis. However, not all animals consistently
displayed increased levels of this subset of T cells, which is
negative for WC1, GD3.1, and GD197 (31, 65). For example,
Fig. 3 shows that a relative increase in
T-cell numbers in the milk of a cow with mastitis is primarily
due to an increase in CD8
cell numbers. Furthermore, the
increase in CD8+ T-cell numbers in this animal is due
primarily to an increase in T-cell numbers, consistent with
previous reports that the predominant T cells in bovine milk are
CD8+ T cells (41, 56). In any case, our studies
do show that different and T-cell subsets are recruited
to the udder, depending on the mastitis pathogen and the host immune
status.
|
Effect of mastitis on neutrophil and lymphocyte adhesion molecule
expression.
One of the earliest events observed after leukocyte
priming or activation is a change in the level of cell surface adhesion molecule expression (4, 20, 27, 28, 30). Therefore, we
analyzed how mastitis affected the level of expression of two adhesion
molecules known to be sensitive indicators of cell activation: L-selectin and CD18. Using flow cytometric analysis, we found that
mastitis infection caused a significant down-regulation of L-selectin
expression on blood lymphocytes and neutrophils (Fig. 4). In milk samples from healthy cows,
L-selectin expression was down-regulated on both lymphocytes and
neutrophils compared to the levels of these cells in blood samples
(Fig. 4), consistent with previous studies showing that L-selectin is
shed during exudation (30). Interestingly, lymphocytes and
neutrophils in the milk of cows with mastitis expressed levels of
L-selectin similar to those isolated from healthy cows, indicating that
maximal down-regulation of L-selectin may have occurred in the blood
and/or during migration from the blood into the udder (Fig. 4).
Analysis of CD18, the common subunit of the 2
integrins (55), showed that CD18 was up-regulated on
lymphocytes and neutrophils from the blood and milk of cows with
mastitis compared to cells from the blood and milk of healthy cows
(Fig. 4).
|
T cells (except for possibly the small number of
CD8+ T cells) would have been missed. Therefore, we
used three-color flow cytometric analysis to further investigate this
issue. As shown in Fig. 5, a
subpopulation of both and T cells stained positively for
L-selectin (~5 to 10%). In addition, the higher level of L-selectin
staining on T cells versus T cells confirms the results
of previous studies by Walcheck and Jutila (61), who
reported that bovine T cells express L-selectin at higher levels
than T cells. In any case, our results clearly show L-selectin
staining on milk leukocytes.
|
T cells (Fig. 5), while little L-selectin
down-regulation was observed for T cells. Consistent with this
observation, the down-regulation of L-selectin on blood lymphocytes
from mastitic cows was also due primarily to the down-regulation of
L-selectin on T cells rather than T cells (data not shown).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The interaction between invading bacteria and the host immune system is a key factor in determining the outcome of an infection (29). Consequently, an effective immune response against any pathogen requires the migration of various sets of myeloid and lymphoid cells into the tissues during the inflammatory response (19). An essential component of the defense of the bovine udder against infection involves the recruitment of large numbers of neutrophils (40); however, it is clear that lymphoid cells are also involved in this process. Currently, little is known about the types of lymphoid cells recruited to the udder during mastitis. Therefore, to better understand the host defense process in mastitis, we have characterized the changes in lymphocyte subset populations present in the blood and milk of cows with naturally occurring staphylococcal and streptococcal mastitis.
The distributions of lymphocyte subsets were similar in blood samples
obtained from cows with mastitis due to naturally occurring staphylococcal and streptococcal infections. However, in both cases, we
observed a significant increase in the total number of T cells
in the blood of infected animals compared to the blood of healthy
animals. In milk samples from infected animals, we observed even larger
increases in the numbers of and T cells. In samples
obtained from cows with staphylococcal mastitis, the increase in
T-cell numbers was due primarily to an increase in CD4+
T-cell numbers. In contrast, the increase in T-cell numbers in
milk from cows with streptococcal mastitis appeared to be due to
parallel increases in both CD4+ and CD8+ T-cell
numbers. This difference in responding T-cell subsets is most
likely due to the nature of the toxins released by staphylococcal and
streptococcal bacteria during the acute stages of infection (44,
59). Indeed, recent studies by Ferens et al. (11)
showed that treatment of bovine peripheral blood mononuclear cell
cultures with staphylococcal enterotoxin C1 (SEC1) led to preferential activation and proliferation of CD4+ T cells. In addition,
they found that activation of CD4+ and CD8+ T
cells by SEC1 was significantly influenced by the proportion of
T cells present in the cultures and suggested that the T-cell/ T-cell ratio might play a role in modulating the immune response to SEC1 (11). In support of this hypothesis, our
present studies demonstrate a significant increase in the
T-cell/ T-cell ratio during naturally occurring mastitis infections.
The mechanisms of antigen recognition and stimulatory signals involved
in the activation and proliferation of T cells are not yet
clearly defined (24). In humans and rodents, T cells represent a minor T-cell population. In contrast, T cells
represent the predominant T-cell population in the circulation of
newborn ruminants (16, 62). Despite the variable numbers of
circulating T cells in different species, recent data suggest
that these cells play an important role in the initial host response to
infectious agents (8). It has been proposed that T
cells complement T cells during the host defense process by
providing a rapid response before the T-cell response has fully
developed, i.e., "the first line of defense" (8, 64).
T cells have been shown to respond to antigen in the context of
major histocompatibility complex molecules; however, most of these
cells do not always require antigen processing to recognize bacterial
antigens (3, 5, 32, 48). Interestingly, some activated
T cells also express high levels of major histocompatibility
complex class II molecules on their surface and are able to present
antigen to CD4+ T cells (6) and prime bacterial
antigen-specific CD8+ T cells (39). These cells
also appear to produce costimulatory molecules as well as cytokines,
demonstrating that T cells do indeed have the capability of
influencing T-cell function (6). Thus, the presence
of increased levels of T cells in milk obtained from cows with
mastitis is consistent with their putative role in modulating the
inflammatory response.
Although T cells have been shown to play a role in contributing
to the inflammatory response (54, 68), recent studies by
several groups have suggested that T cells may have a protective or anti-inflammatory function (10, 35, 36, 47). For example, TCR gene knockout mice infected with Mycobacterium
tuberculosis were able to control early infection in a manner
similar to wild-type mice; however, a substantial pyogranulomatous
response was observed in the knockout mice but not in the wild-type
controls (10). These authors concluded that T cells
do not directly protect against infection but instead play a role in
modulating local cellular traffic by promoting the influx of
lymphocytes and monocytes and limiting the access of inflammatory cells
that do not contribute to protection but can cause tissue damage
(10). In support of this idea, Mukasa et al. (35)
found that depletion of T cells accelerated testicular
inflammation in mice injected with Listeria monocytogenes.
In addition, studies by Park et al. (42, 43) showed that a
subset of CD8+ T cells in bovine milk was
responsible for the down-regulation of the response of CD4+
T cells to staphylococcal antigens. Finally, T cells have also
been found to produce growth factors that may play a role in the
healing of epithelia damaged by infection or by inflammation (2,
22). Thus, it is clear that T cells do play a role in
modulating the inflammatory response; however, it is currently not
known whether the primary role of increased levels of T cells is
to regulate the magnitude of the immune response or to directly
contribute to host tissue protection. Studies are in progress to
investigate this issue.
Several different T-cell subsets have been identified, as
defined by their respective TCR expression (18, 66), and recent studies have demonstrated that the distinct T-cell
subsets localize to specific tissues. For example, murine T
cells homing to the intestinal epithelium, skin, vagina, uterus, and
tongue utilize a distinct TCR (17). Recently, Wilson
et al. (65) showed that the CD8+
CD2+ T-cell subset exhibited a defined tissue
tropism for the spleen but did not accumulate efficiently at sites of
inflammation. In addition, Wilson et al. (66) described
three novel anti-bovine TCR antibodies (GD3.8, GD197, and GD3.1)
and showed that these antibodies could be used to define a distinct
T-cell subset that preferentially localized in inflamed lymph
node tissue. Specifically, GD3.1+ T cells were found
to be preferentially enriched at the site of inflammation
(66). The data presented here support this observation, as
we found that the increase in T-cell numbers in milk from cows
with acute mastitis was also primarily due to a preferential increase
in GD3.1+ T-cell numbers.
The recruitment of leukocytes is one of the first steps in the host
response to infection, and leukocyte emigration from the blood to sites
of inflammation involves a sequential interaction of adhesion molecules
expressed by leukocytes and endothelial cells (1, 13). Two
groups of adhesion molecules known to play important roles in this
process are the selectins, which mediate leukocyte rolling on the
endothelium (reviewed in reference 23), and the
2 integrins, which mediate tight adhesion and diapedesis
(reviewed in reference 12). Both of these groups of
adhesion molecules have been found to be sensitive indicators of
leukocyte activation (20, 27). Depending on the treatment, neutrophil priming or activation can cause the shedding of L-selectin from the cell surface or the up-regulation of CD11b/CD18 or both (4, 7, 20, 27). Recently, it has also been shown that the
exudation of human neutrophils into skin chambers in vivo causes the
shedding of L-selectin and the modest up-regulation of CD11b/CD18
(30, 50). In the present studies, we found that lymphocytes
and neutrophils from the milk of healthy cows expressed significantly
lower levels of L-selectin than cells obtained from the blood of these
cows; however, we observed very little up-regulation of CD18 in milk
leukocytes compared to blood leukocytes. In contrast, lymphocytes and
neutrophils obtained from the milk of cows with mastitis exhibited
significant up-regulation of CD18, consistent with an activated state
due to the presence of bacterial pathogens. Since these cells already
down-regulated L-selectin during migration into the udder, little or no
further down-regulation or shedding of L-selectin was observed in milk
leukocytes isolated from cows with mastitis. In support of these
findings, a similar decrease in L-selectin expression has been observed
for caprine milk leukocytes (14). Interestingly, we observed
the down-regulation or shedding of L-selectin and the up-regulation of
CD18 in blood lymphocytes and neutrophils obtained from cows with
mastitis compared to blood leukocytes obtained from healthy cows. Thus,
changes in the expression of these adhesion molecules on blood
leukocytes might represent a potential diagnostic indicator of mastitis
that could be easily and rapidly evaluated.
The changes in adhesion molecule expression observed on cells from cows
with mastitis reflect the overall host response to infection, where
increased adhesion mediated through the 2 integrins would facilitate adherence to the pathogen, phagocytosis, and killing
(12). Enhanced expression of 2 integrins
would also facilitate leukocyte migratory potential through increased
adhesive interactions with the endothelium, i.e., more effective
transition from rolling to adherent cells. Shedding of L-selectin
occurs during the process of diapedesis and may be required to
dissociate the cells from ligands associated with the transmigration
process and induce unresponsiveness to extracellular ligands
(58). The role of L-selectin shedding by cells in the blood
is not yet clear. One possibility is that soluble L-selectin plays a
cytokine-like role in priming or even stimulating the host defense
response. Soluble forms of L-selectin have been detected in the plasma
of patients at risk for adult respiratory distress syndrome, and a
correlation between reduced levels of soluble L-selectin and progression to this syndrome has been observed (9). In
addition, recent studies by Ruchaud-Sparagano and coworkers
(46) showed that soluble E-selectin exerted a
proinflammatory effect on neutrophil function. Another possible
function of L-selectin shedding by blood leukocytes is to limit the
number of leukocytes accumulating at sites of inflammation and,
thereby, to limit excessive tissue injury caused by these inflammatory
cells. This idea is supported by the studies of McGill et al.
(33), who reported that intravascular shedding of L-selectin
might be a mechanism for controlling neutrophil exudation in patients
with systemic inflammatory response syndrome. It has also been proposed
that intravascular L-selectin shedding may be a general mechanism used
by the human body to control inflammation, thus explaining the normal
neutrophilia observed during inflammatory disease (45). In a
similar manner, the intravascular shedding of L-selectin by leukocytes
in mastitic cows may be a mechanism for controlling the level of
leukocyte accumulation in the mammary gland and preventing further
tissue damage due to inflammation.
In summary, we have characterized lymphocyte subset distributions and
adhesion molecule expression on leukocytes from the blood and milk of
healthy cows and cows with naturally acquired mastitis of
staphylococcal and streptococcal origin. Our studies show that,
depending on the type of mastitis pathogen, differential T-cell subset
recruitment to the udder occurs. Both and T cells were
recruited to the mammary gland; however, depending on the infectious
agent, various ratios of CD4+ and CD8+ T
cells and distinct T-cell subsets were found in the milk. In
addition, the shedding of L-selectin and the up-regulation of CD18 by
leukocytes in the blood may provide a sensitive indicator of early
inflammatory responses during bovine mastitis. Further studies are
necessary to define the specific roles of the various leukocyte subsets
in the host immune response as well as the contributions of the
pathogens involved. Understanding the role of specific T-cell subsets
in mastitis may have a significant impact on the development of
effective treatments or vaccines.
| |
ACKNOWLEDGMENTS |
|---|
We thank Mark Jutila and Eric Wilson (Montana State University, Bozeman) for generously providing antibodies for this research and for reviewing the manuscript. We also thank David Bos and Marilyn Bos (Faith Dairy, Bozeman, Mont.) for allowing us to study cows from their dairy herd.
This work was supported in part by USDA/NRICGP grant 9502274 (to M.T.Q.), USDA/NRICGP grant 9903508 (to J.S.), NSF equipment grant DBI-9604797 (to M.T.Q.), NIH equipment grant S10 RR11877, an equipment grant from the M. J. Murdock Charitable Trust, USDA Animal Health Formula Funds, and the Montana State University Agricultural Experimental Station. Mark T. Quinn is an Established Investigator of the American Heart Association.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717. Phone: (406) 994-5721. Fax: (406) 994-4303. E-mail: mquinn{at}montana.edu.
Editor: J. R. McGhee
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Ali, H., B. Haribabu, R. M. Richardson, and R. Snyderman. 1997. Mechanisms of inflammation and leukocyte activation. Med. Clin. N. Am. 81:1-28[Medline]. |
| 2. |
Boismenu, R., and W. L. Havran.
1994.
Modulation of epithelial cell growth by intraepithelial T cells.
Science
266:1253-1255 |
| 3. | Boismenu, R., and W. L. Havran. 1997. An innate view of gamma delta T cells. Curr. Opin. Immunol. 9:57-63[Medline]. |
| 4. | Borregaard, N., L. Kjeldsen, H. Sengelov, M. S. Diamond, T. A. Springer, H. C. Anderson, T. K. Kishimoto, and D. F. Bainton. 1994. Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J. Leukoc. Biol. 56:80-87[Abstract]. |
| 5. | Chien, Y. H., R. Jores, and M. P. Crowley. 1996. Recognition by gamma/delta T cells. Annu. Rev. Immunol. 14:511-532[Medline]. |
| 6. |
Collins, R. A.,
D. Werling,
S. E. Duggan,
A. P. Bland,
K. R. Parsons, and C. J. Howard.
1998.
T cells present antigen to CD4+ T cells.
J. Leukoc. Biol.
63:707-714[Abstract].
|
| 7. | Condliffe, A. M., E. R. Chilvers, C. Haslett, and I. Dransfield. 1996. Priming differentially regulates neutrophil adhesion molecule expression/function. Immunology 89:105-111[Medline]. |
| 8. |
De Libero, G.
1997.
Sentinel function of broadly reactive human T cells.
Immunol. Today
18:22-26[Medline].
|
| 9. | Donnelly, S. C., C. Haslett, I. Dransfield, C. E. Robertson, D. C. Carter, J. A. Ross, I. S. Grant, and T. F. Tedder. 1994. Role of selectins in development of adult respiratory distress syndrome. Lancet 344:215-219[Medline]. |
| 10. |
D'Souza, C. D.,
A. M. Cooper,
A. A. Frank,
R. J. Mazzaccaro,
B. R. Bloom, and I. M. Orme.
1997.
An anti-inflammatory role for T lymphocytes in acquired immunity to Mycobacterium tuberculosis.
J. Immunol.
158:1217-1221[Abstract].
|
| 11. |
Ferens, W. A.,
W. C. Davis,
M. J. Hamilton,
Y. H. Park,
C. F. Deobald,
L. Fox, and G. Bohach.
1998.
Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C.
Infect. Immun.
66:573-580 |
| 12. |
Gahmberg, C. G.,
M. Tolvanen, and P. Kotovuori.
1997.
Leukocyte adhesion. Structure and function of human leukocyte 2-integrins and their cellular ligands.
Eur. J. Biochem.
245:215-232[Medline].
|
| 13. | Gahmberg, C. G., L. Valmu, S. Fagerholm, P. Kotovuori, E. Ihanus, L. Tian, and T. Pessa-Morikawa. 1998. Leukocyte integrins and inflammation. Cell. Mol. Life Sci. 54:549-555[Medline]. |
| 14. | Guiguen, F., T. Greenland, E. Pardo, G. Panaye, and J. F. Mornex. 1996. Flow cytometric analysis of goat milk lymphocytes: subpopulations and adhesion molecule expression. Vet. Immunol. Immunopathol. 53:173-178[Medline]. |
| 15. | Harmon, R. J. 1994. Physiology of mastitis and factors affecting somatic cell counts. J. Dairy Sci. 77:2103-2112[Abstract]. |
| 16. |
Hein, W. R., and C. R. Mackay.
1991.
Prominence of T cells in the ruminant immune system.
Immunol. Today
12:30-34[Medline].
|
| 17. |
Itohara, S.,
A. G. Farr,
J. J. Lafaille,
M. Bonneville,
Y. Takagaki,
W. Haas, and S. Tonegawa.
1990.
Homing of a thymocyte subset with homogeneous T-cell receptors to mucosal epithelia.
Nature
343:754-757[Medline].
|
| 18. |
Jones, W. M.,
B. Walcheck, and M. A. Jutila.
1996.
Generation of a new T cell-specific monoclonal antibody (GD3.5). Biochemical comparison of GD3.5 antigen with the previously described workshop cluster 1 (WC1) family.
J. Immunol.
156:3772-3779[Abstract].
|
| 19. |
Jutila, M. A.
1999.
Recruitment of / T-cells and other T-cell subsets to sites of inflammation, p. 193-214.
In
C. N. Serhan, and P. A. Ward (ed.), Molecular and cellular basis of inflammation. Humana Press, Inc., Totowa, N.J.
|
| 20. | Jutila, M. A., L. Rott, E. L. Berg, and E. C. Butcher. 1989. Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1. J. Immunol. 143:3318-3324[Abstract]. |
| 21. |
Jutila, M. A.,
G. Watts,
B. Walcheck, and G. S. Kansas.
1992.
Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin.
J. Exp. Med.
175:1565-1573 |
| 22. |
Kagnoff, M. F.
1998.
Current concepts in mucosal immunity. III. Ontogeny and function of T cells in the intestine.
Am. J. Physiol.
274:G455-G458 |
| 23. |
Kansas, G. S.
1996.
Selectins and their ligands: current concepts and controversies.
Blood
88:3259-3287 |
| 24. |
Kaufmann, S. H.
1996.
and other unconventional T lymphocytes: what do they see and what do they do?
Proc. Natl. Acad. Sci. USA
93:2272-2279 |
| 25. | Keefe, G. P. 1997. Streptococcus agalactiae mastitis: a review. Can. Vet. J. 38:429-437[Medline]. |
| 26. | Kehrli, M. E., Jr., and D. E. Shuster. 1994. Factors affecting milk somatic cells and their role in health of the bovine mammary gland. J. Dairy Res. 77:619-627. |
| 27. |
Kishimoto, T. K.,
M. A. Jutila,
E. L. Berg, and E. C. Butcher.
1989.
Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science
245:1238-1241 |
| 28. |
Kishimoto, T. K.,
M. A. Jutila, and E. C. Butcher.
1990.
Identification of a human peripheral lymph node homing receptor: a rapidly down-regulated adhesion molecule.
Proc. Natl. Acad. Sci. USA
87:2244-2248 |
| 29. | Kotwal, G. J. 1997. Microorganisms and their interaction with the immune system. J. Leukoc. Biol. 62:415-429[Abstract]. |
| 30. | Kuhns, D. B., D. A. L. Priel, and J. I. Gallin. 1995. Loss of L-selectin (CD62L) on human neutrophils following exudation in vivo. Cell. Immunol. 164:306-310[Medline]. |
| 31. |
MacHugh, N. D.,
J. K. Mburu,
M. J. Carol,
C. R. Wyatt,
J. A. Orden, and W. C. Davis.
1997.
Identification of two distinct subsets of bovine T cells with unique cell surface phenotype and tissue distribution.
Immunology
92:340-345[Medline].
|
| 32. | Matis, L. 1991. Specificity and selection of gamma-delta receptor-expressing T cells. Immunol. Res. 10:5-14[Medline]. |
| 33. |
McGill, S. N.,
N. A. Ahmed,
F. Hu,
R. P. Michel, and N. V. Christou.
1996.
Shedding of L-selectin as a mechanism for reduced polymorphonuclear neutrophil exudation in patients with the systemic inflammatory response syndrome.
Arch. Surg.
131:1141-1146 |
| 34. | McMichael, A. J., F. M. Gotch, and J. E. Hildreth. 1982. Lysis of allogeneic human lymphocytes by nonspecifically activated T-like cells. Eur. J. Immunol. 12:1002-1005[Medline]. |
| 35. |
Mukasa, A.,
K. Hiromatsu,
G. Matsuzaki,
R. O'Brien,
W. Born, and K. Nomoto.
1995.
Bacterial infection of the testis leading to autoaggressive immunity triggers apparently opposed responses of and T cells.
J. Immunol.
155:2047-2056[Abstract].
|
| 36. |
Mukasa, A.,
M. Lahn,
E. K. Pflum,
W. Born, and R. L. O'Brien.
1997.
Evidence that the same T cells respond during infection-induced and autoimmune inflammation.
J. Immunol.
159:5787-5794[Abstract].
|
| 37. | Myllys, V., J. Ridell, J. Björkroth, I. Biese, and S. Pyörälä. 1997. Persistence in bovine mastitis of Staphylococcus aureus clones as assessed by random amplified polymorphic DNA analysis, ribotyping and biotyping. Vet. Microbiol. 57:245-251[Medline]. |
| 38. | Naessens, J., C. J. Howard, and J. Hopkins. 1997. Nomenclature and characterization of leukocyte differentiation antigens in ruminants. Immunol. Today 18:365-368[Medline]. |
| 39. |
Nomura, A.,
G. Matsuzaki,
H. Takada,
K. Hiromatsu,
S. Nabeshima,
T. Nakamura,
K. Kishihara, and K. Nomoto.
1998.
The role of T cells in induction of bacterial antigen-specific protective CD8+ cytotoxic T cells in immune response against the intracellular bacteria Listeria monocytogenes.
Immunology
95:226-233[Medline].
|
| 40. |
Paape, M. J.,
W. P. Wergin,
A. J. Guidry, and R. E. Pearson.
1979.
Leukocytes second line of defense against invading mastitis pathogens.
J. Dairy Sci.
62:135-153.
|
| 41. | Park, Y. H., L. K. Fox, M. J. Hamilton, and W. C. Davis. 1992. Bovine mononuclear leukocyte subpopulations in peripheral blood and mammary gland secretions during lactation. J. Dairy Sci. 75:998-1006[Abstract]. |
| 42. | Park, Y. H., L. K. Fox, M. J. Hamilton, and W. C. Davis. 1993. Suppression of proliferative response of BoCD4+ T lymphocytes by activated BoCD8+ T lymphocytes in the mammary gland of cows with Staphylococcus aureus mastitis. Vet. Immunol. Immunopathol. 36:137-151[Medline]. |
| 43. |
Park, Y. H.,
S. C. Jung,
J. S. Moon,
B. G. Ku,
C. R. Wyatt,
M. J. Hamilton,
L. K. Fox, and W. C. Davis.
1994.
A subset of mammary gland T lymphocytes downregulates BoCD4 T lymphocyte response to Staphylococcus aureus in cattle with intramammary infection.
Korean J. Immunol.
16:19-27.
|
| 44. | Rago, J. V., and P. M. Schlievert. 1998. Mechanisms of pathogenesis of staphylococcal and streptococcal superantigens. Curr. Top. Microbiol. Immunol. 225:81-97[Medline]. |
| 45. | Rogowski, O., Y. Sasson, M. Kassirer, D. Zeltser, N. Maharshak, N. Arber, P. Halperin, J. Serrov, P. Sorkin, A. Eldor, and S. Berliner. 1998. Down-regulation of the CD62L antigen as a possible mechanism for neutrophilia during inflammation. Br. J. Haematol. 101:666-669[Medline]. |
| 46. | Ruchaud-Sparagano, M. H., E. M. Drost, S. C. Donnelly, M. I. Bird, C. Haslett, and I. Dransfield. 1998. Potential pro-inflammatory effects of soluble E-selectin upon neutrophil function. Eur. J. Immunol. 28:80-89[Medline]. |
| 47. |
Saunders, B. M.,
A. A. Frank,
A. M. Cooper, and I. M. Orme.
1998.
Role of gamma delta T cells in immunopathology of pulmonary Mycobacterium avium infection in mice.
Infect. Immun.
66:5508-5514 |
| 48. | Schild, H., N. Mavaddat, C. Litzenberger, E. W. Ehrich, M. M. Davis, J. A. Bluestone, L. Matis, R. K. Draper, and Y. H. Chien. 1994. The nature of major histocompatibility complex recognition by gamma delta T cells. Cell 76:29-37[Medline]. |
| 49. | Schmaltz, R., B. Bhogal, J. Wang, Y. Y. Wang, C. R. Mackay, S. S. Chen, and L. Larson. 1996. Characterisation of leucocytic somatic cells in bovine milk. Res. Vet. Sci. 61:179-181[Medline]. |
| 50. | Sengelov, H., P. Follin, L. Kjeldsen, K. Lollike, C. Dahlgren, and N. Borregaard. 1995. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J. Immunol. 154:4157-4165[Abstract]. |
| 51. | Sipes, K. M., H. Edens, M. E. Kehrli, Jr., J. E. Cutler, H. M. Miettinen, M. A. Jutila, and M. T. Quinn. 1999. Analysis of surface antigen expression and host defense function in leukocytes from calves with bovine leukocyte adhesion deficiency: comparison of heterozygous and homozygous animals. Am. J. Vet. Res. 60:1255-1261[Medline]. |
| 52. | Soltys, J., S. D. Swain, K. M. Sipes, L. K. Nelson, M. A. Jutila, and M. T. Quinn. 1999. Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods. J. Immunol. Methods 226:71-84[Medline]. |
| 53. | Sordillo, L. M., K. Shafer-Weaver, and D. DeRosa. 1997. Immunobiology of the mammary gland. J. Dairy Sci. 80:1851-1865[Abstract]. |
| 54. |
Spinozzi, F.,
E. Agea,
O. Bistoni,
N. Forenza, and A. Bertotto.
1998.
T cells, allergen recognition and airway inflammation.
Immunol. Today
19:22-26[Medline].
|
| 55. | Springer, T. A. 1990. Adhesion receptors of the immune system. Nature 346:425-434[Medline]. |
| 56. | Taylor, B. C., J. D. Dellinger, J. S. Cullor, and J. L. Stott. 1994. Bovine milk lymphocytes display the phenotype of memory T cells and are predominantly CD8+. Cell. Immunol. 156:245-253[Medline]. |
| 57. | Taylor, B. C., R. G. Keefe, J. D. Dellinger, Y. Nakamura, J. S. Cullor, and J. L. Stott. 1997. T cell populations and cytokine expression in milk derived from normal and bacteria-infected bovine mammary glands. Cell. Immunol. 182:68-76[Medline]. |
| 58. | Tedder, T. F., D. A. Steeber, A. Chen, and P. Engel. 1995. The selectins: vascular adhesion molecules. FASEB J. 9:866-873[Abstract]. |
| 59. | Torres, B. A., and H. M. Johnson. 1998. Modulation of disease by superantigens. Curr. Opin. Immunol. 10:465-470[Medline]. |
| 60. | Villanueva, M. R., J. W. Tyler, and M. C. Thurmond. 1991. Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk. J. Am. Vet. Med. Assoc. 198:1398-1400[Medline]. |
| 61. |
Walcheck, B., and M. A. Jutila.
1994.
Bovine T cells express high levels of functional peripheral lymph node homing receptor (L-selectin).
Int. Immunol.
6:81-91 |
| 62. |
Walcheck, B.,
G. Watts, and M. A. Jutila.
1993.
Bovine / T cells bind E-selectin via a novel glycoprotein receptor: first characterization of a lymphocyte/E-selectin interaction in an animal model.
J. Exp. Med.
178:853-863 |
| 63. | Walcheck, B., M. White, S. Kurk, T. K. Kishimoto, and M. A. Jutila. 1992. Characterization of the bovine peripheral lymph node homing receptor: a lectin cell adhesion molecule (LECAM). Eur. J. Immunol. 22:469-476[Medline]. |
| 64. |
Williams, N.
1998.
T cells on the mucosal frontline.
Science
280:198-200 |
| 65. |
Wilson, E.,
M. K. Aydintug, and M. A. Jutila.
1999.
A circulating bovine T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin.
J. Immunol.
162:4914-4919 |
| 66. |
Wilson, E.,
B. Walcheck,
W. C. Davis, and M. A. Jutila.
1998.
Preferential tissue localization of bovine T cell subsets defined by anti-T cell receptor for antigen antibodies.
Immunol. Lett.
64:39-44[Medline].
|
| 67. | Yancey, R. J. J. 1999. Vaccines and diagnostic methods for bovine mastitis: fact and fiction. Adv. Vet. Med. 41:257-273[Medline]. |
| 68. |
Zuany-Amorim, C.,
C. Ruffie,
S. Haile,
B. B. Vargaftig,
P. Pereira, and M. Pretolani.
1998.
Requirement for T cells in allergic airway inflammation.
Science
280:1265-1267 |
Infection and Immunity, December 1999, p. 6293-6302, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Trigo, G., Dinis, M., Franca, A., Bonifacio Andrade, E., Gil da Costa, R. M., Ferreira, P., Tavares, D.
(2009). Leukocyte populations and cytokine expression in the mammary gland in a mouse model of Streptococcus agalactiae mastitis. J Med Microbiol
58: 951-958
[Abstract] [Full Text] -
Seo, K. S., Park, J. Y., Davis, W. C., Fox, L. K., McGuire, M. A., Park, Y. H., Bohach, G. A.
(2009). Superantigen-mediated differentiation of bovine monocytes into dendritic cells. J. Leukoc. Biol.
85: 606-616
[Abstract] [Full Text] -
Moreto, M., Perez-Bosque, A.
(2009). Dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa. J ANIM SCI
87: E92-E100
[Abstract] [Full Text] -
Mehrzad, J., Janssen, D., Duchateau, L., Burvenich, C.
(2008). Increase in Escherichia coli Inoculum Dose Accelerates CD8+ T-Cell Trafficking in the Primiparous Bovine Mammary Gland. J DAIRY SCI
91: 193-201
[Abstract] [Full Text] -
Nofrarias, M., Manzanilla, E. G., Pujols, J., Gibert, X., Majo, N., Segales, J., Gasa, J.
(2006). Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs. J ANIM SCI
84: 2735-2742
[Abstract] [Full Text] -
Michie, C, Lockie, F, Lynn, W
(2003). The challenge of mastitis. Arch. Dis. Child.
88: 818-821
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»