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Immunology Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Selectins, integrins, and chemotactic factors show a close relationship
with cytoskeleton and exert an important role as signal-transducing
receptors, activating different biochemical pathways, including the
mitogen-activated protein kinase
(MAPK)3 cascade. It
has been reported that the cross-linking of L-selectin on neutrophils
(3), the exposure of neutrophils to TNF-
or LPS
(4, 5), or stimulation of neutrophils by chemotactic
factors such as fMLP (6) result in the phosphorylation and
activation of a p38 MAPK. Consistent with a role for p38 MAPK in
neutrophil recruitment have been in vivo studies showing that p38 MAPK
inhibitors reduce the neutrophil infiltration in numerous inflammatory
processes. Neutrophil infiltration into the peritoneal cavity induced
by arachidonic acid (7), as well as carrageenan or urate
crystals (8), was inhibited by SKF86002. In other p38 MAPK
inhibition studies, infiltration of neutrophils into the airspace and
lung induced by LPS (9) in Clostridium
difficile toxin A-induced enteritis (10) and in
Helicobacter pylori-induced gastritis (11) was
inhibited by M39, SB203580, and FR167653, respectively.
Although these studies suggest the anti-inflammatory potential of the p38 MAPK inhibitors, they do not provide insight as to the specific mechanism(s) of action. For example, in vitro p38 MAPK inhibitors have been proposed as inhibitors of selectin function (12), integrin function as it pertains to adhesion (13), as well as chemotaxis (6), suggesting that the p38 MAPK inhibitors are general inhibitors of the recruitment cascade. However, this has never been systematically examined in the in vivo setting.
Therefore, in this study we investigated the role of p38 MAPK in leukocyte recruitment in vivo. Using intravital microscopy and a novel in vivo chemotaxis assay system, we were able to visualize the leukocyte rolling, adhesion, emigration, and directional migration toward a source of chemotactic factor in the extravascular tissue of the cremaster muscle. The latter was performed with time-lapse photography. The data reveal absolutely no effect of SB203580 and SKF86002 on selectin-dependent leukocyte rolling or integrin-dependent leukocyte adhesion, but the data do show very significant effects upon downstream events of neutrophil adhesion, i.e., a profound reduction in neutrophil emigration and chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). All mice weighed between 20 and 30 g and were used between 6 and 10 wk of age. The animals were anesthetized with an i.p. injection of a mixture of 10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and 200 mg/kg ketamine hydrochloride (Rogar/STB, Montreal, Quebec, Canada). For all protocols, the left jugular vein was cannulated to administer additional anesthetic or drugs if necessary.
Intravital microscopy
The mouse cremaster preparation was used to study the behavior of leukocytes in the microcirculation and adjacent connective tissue as previously described (14). Briefly, an incision was made in the scrotal skin to expose the left cremaster muscle, which was then carefully removed from the associated fascia. A lengthwise incision was made on the ventral surface of the cremaster muscle using a cautery. The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity. The muscle was then spread out over an optically clear viewing pedestal and was secured along the edges with 4–0 suture. The exposed tissue was superfused with warm bicarbonate-buffered saline (pH 7.4). An intravital microscope (Axiolskip; Carl Zeiss Canada, Don Mills, Ontario, Canada) with a x25 objective lens (Weltzlar L25/o.35; E. Leitz Inc., Munich, Germany), and a x10 eyepiece was used to examine the cremasteric microcirculation. A video camera (5100 HS; Panasonic, Osaka, Japan) was used to project the images onto a monitor, and the images were recorded for playback analysis using a conventional videocassette recorder or time-lapse videocassette recorder.
Single unbranched cremasteric venules (25–40 µm in diameter) were selected, and to minimize variability, the same section of cremasteric venule was observed throughout the experiment. The number of rolling, adherent, and emigrated leukocytes was determined offline during video playback analysis. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a given vessel. The flux of rolling cells was measured as the number of rolling cells passing by a given point in the venule per minute. A leukocyte was considered to be adherent if it remained stationary for at least 30 s, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-µm length of venule. Leukocyte emigration was defined as the number of cells in the extravascular space within 200 x 300 µm area, as well as in each 50 µm of distance from the venule. Only cells adjacent to and clearly outside the vessel under study were counted as emigrated. Directional migration or chemotaxis was examined using time-lapse photography and was plotted as a function of distance moved toward the chemotactic source.
Induction of chemotaxis
An agarose gel containing keratinocyte-derived cytokine (KC),
the murine homolog of GRO- (R&D Systems, Minneapolis, MN), was used
to induce chemotaxis in the cremaster preparation (15).
The agarose gel was prepared adding 10 ml of 2x HBSS to a boiling
concentrated agarose solution (4% in 10 ml of distilled water). A
100-µl aliquot of this solution was removed and KC was added to this
aliquot to achieve a final concentration of 5.2 µM. To enable
visualization of the gel on the cremaster muscle, a small amount of
india ink was added to each preparation. A 1-mm3
piece of the mixture was punched out using the tip of a Pasteur
pipette. This piece of gel was carefully placed on the surface of the
cremaster in a preselected avascular area 350 µm (two monitor screens
wide) from a postcapillary venule. The gel was held in place using a
coverslip, and the tissue was superfused beneath the coverslip at a
minimum rate (0.7 ml/min) so as not to disrupt the chemotactic gradient
established adjacent to the agarose pellet. The image was recorded for
90 min: 30 min for control without gel (in presence of drugs/vehicle)
and 60 min with gel. In some experiments, only the gel (without KC) was
placed on the surface on the cremaster.
Inhibition of p38 MAPK activity
Thirty minutes before administration of KC, the animals were treated with two different p38 MAPK inhibitors: SKF860002 or SB203580. SKF86002 (Calbiochem, La Jolla, CA) was given i.v. in different doses (5, 10, and 20 mg/kg) or locally by superfusion (0.7 mM). SB203580 (AG Scientific, San Diego, CA) was given i.v. in a dose of 20 mg/kg based on previously reported optimal concentrations (16, 17).
Assay for p38 MAPK phosphorylation and activity in cremaster muscle
After the intravital microscopy experiments were completed, the
cremaster muscles were removed and immediately frozen at -70°C. The
cremaster muscles were incubated in 0.5 ml of ice-cold cell lysis
buffer plus 1 mM PMSF for 5 min. The lysis buffer contained 20 mM Tris,
pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1 mM
Na3VO4, 1% Triton X-100,
2.5 mM sodium pyrophosphate, 1 mM 
-glycerolphosphate, and 1 µg/ml
leupeptin. The tissues were sonicated four times for 5 s each on
ice, and were centrifuged for 10 min at 4°C. The supernatant was
stored at -70°C for activity assay.
For immunoprecipitation of phosphorylated p38 MAPK, 200 µl of cell
lysate containing 
200 µg of total protein was incubated with 20
µl of resuspended immobilized Phospho-p38 MAPK
(Thr180/Tyr182) monoclonal Ab (New England
Biolabs, Beverly, MA) overnight at 4°C. The samples were then
centrifuged for 30 s at 4°C, and the pellet was washed twice
with 500 µl of lysis buffer and washed twice with 500 µl of kinase
buffer containing 20 mM Tris (pH 7.5), 5 mM -glycerolphosphate, 2 mM
DTT, 0.1 mM Na3VO4, and 10
mM MgCl2. Immune complexes were resuspended in 50
µl of kinase buffer supplemented with 200 µM ATP and 2 µg of
activating transcription factor-2 (ATF-2, New England Biolabs) for 30
min at 30°C. The reaction was terminated with 25 µl of 3x SDS
sample buffer containing 187.5 mM Tris-HCL (pH 6.8 at 25°C), 6%
(w/v) SDS, 30% glycerol, 150 mM DTT, and 0.03% (w/v) bromphenol blue.
The samples were boiled for 5 min and loaded on 10% SDS-PAGE gels.
Gels were transferred onto polyvinylidene difluoride membranes using a
semidry electrophoretic transfer apparatus (Bio-Rad, Hercules, CA) for
55 min at 180 mA. Following transfer, the membranes were incubated for
1 h at room temperature in 25 ml of blocking buffer (2% skim
milk/TBS with 0.1% Tween 20). After washing three times for 5 min each
with 15 ml of TBS with 0.1% Tween 20, the membrane and the primary Ab
were incubated in 10 ml of Ab dilution buffer with gentle agitation
overnight at 4°C. After incubation, the membranes were washed and
incubated with HRP-conjugated anti-rabbit secondary Ab and
HRP-conjugated anti-biotin Ab to detect biotinylated protein
markers in 10 ml of blocking buffer with gentle agitation for 1 h
at room temperature. Then the membranes were washed and incubated with
10 ml of LumiGLO (0.5 ml of 20x LumiGLO, 0.5 ml of 20x peroxide, and
9.0 ml of water) with gentle agitation for 1 min at room temperature.
Finally, the membranes were drained of excess developing solution,
wrapped in plastic wrap, and exposed to x-ray film.
Histology
At the end of each intravital microscopy experiment, the cremaster muscles were removed and fixed in 10% neutral buffered formalin. The tissues were dehydrated gradually in ethanol, embedded in paraffin, cut into 4-µm sections, stained with H&E, and examined under direct light microscopy.
Statistical analysis
The results were expressed as means ± SEM. Student’s t test was applied with a Bonferroni correction where necessary. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
demonstrates the impact of
systemic administration of p38 MAPK inhibitor on the first two steps of
the multistep cascade of leukocyte recruitment (leukocyte rolling and
adhesion). With time, rolling decreases in untreated sham preparations
(just gel without KC). KC administration maintained rolling cells at
100 cells/min. Treatment of animals with p38 MAPK inhibitor did not
affect the number of rolling leukocytes associated with KC (Fig. 1A). KC caused a significant increase in the number of
adherent leukocytes. The p38 MAPK inhibitor (SB203580) had no
inhibitory effect on the number of adherent cells (Fig. 1B).
Simply placing the gel without KC onto the preparation does not induce
any adhesion (sham group).
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The p38 MAPK inhibitor impacted upon downstream events of
leukocyte adhesion. The slow release of KC from the gel induced very
significant emigration in cremaster muscle (Fig. 2A). Without KC (just gel),
fewer than 10 cells are seen within the field of view. The addition of
KC induces emigration of >70 cells/field of view. The cells move
toward the KC source. Intravenous treatment with p38 MAPK inhibitor
(SKF86002) reduced the total number of emigrated cells in a
dose-response fashion. Significance was achieved at 20 mg/kg. Fig. 2B reveals identical results with a second inhibitor of p38
MAPK (SB203580).
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Aside from inhibiting emigration, the i.v. treatments with either
p38 inhibitor impaired the chemotactic response induced by KC. When the
cremaster muscle was divided into 25-µm sections, 20 leukocytes
were within the first 25 µm, 15 leukocytes were in the next 25 µm,
and so on, with fewer and fewer cells available in each subsequent
partition. SB203580 inhibited the number of chemotaxing cells found in
each partition (Fig. 3A). It
was apparent that the further away from the vessel, the greater the
inhibition of leukocytes chemotaxis. For example, a 56–63% inhibition
of the number of leukocytes was seen in the first 75 µm, whereas 83%
inhibition was seen at 75–100 µm and 100% inhibition was noted at
further distances. Similarly, at a dose of 20 mg/kg, SKF86002 inhibited
62% of the chemotaxis in the first 50 µm, 70% in the space from
50–100 µm, and 90% in the space from 100 to 150 µm (Fig. 3B). At lower concentrations of SKF86002, this observation
was even more apparent, with inhibition of chemotaxis becoming apparent
only at 100–150 µm away from the blood vessel.
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Although the data clearly demonstrate that both emigration and
subsequent chemotaxis were affect by the p38 MAPK inhibitors, it could
be argued that fewer emigrated leukocytes beget fewer cells
chemotaxing. To dissociate these two events, the tissue was superfused
with p38 MAPK inhibitor. This still permitted leukocytes to emigrate
out of the vessels. Fig. 4A
highlights that the total number of emigrated cells was quite similar
during p38 MAPK inhibitor superfusion of the tissue. Clearly,
circulating cells within the vasculature were still able to roll,
adhere, and even emigrate before the inhibitor had time to
significantly affect the leukocyte biology. Fig. 4B
demonstrates that once outside the vessel, fewer cells were seen
chemotaxing toward the source of KC. Most cells remained closely
opposed to the blood vessels (0–50 µm) in the presence of
SKF86002.
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Fig. 5A is a
photomicrograph intended to show the dramatic impairment in chemotaxis
and that the cells are neutrophils. The micrograph is not intended to
show emigration. Leukocyte behavior was quantitated using intravital
microscopy (
Figs. 1–4
). The figure shows the presence of emigrated
neutrophils polarized toward the source of KC and away from the venule
in animals not receiving p38 MAPK inhibitor. This histological sample
was taken at 1 h of KC with fewer cells seen away from the vessel at
earlier times (not shown). Fig. 5B is an insert showing that
these cells are polymorphonuclear leukocytes. Fig. 5C shows
a decrease in the total number of neutrophils in the tissue surrounding
the blood vessel, but significant adhesion of cells in the vessel in
p38-treated mice. The arrow indicates the direction toward the KC
source. Although it is difficult to elucidate from this photomicrograph
whether the cells are adherent on the luminal or abluminal side,
intravital microscopy allowed for real time detection of adhesion and
emigration and revealed that following p38 MAPK treatment, the majority
of cells adhered but did not emigrate.
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Fig. 6A shows that the
phosphorylation of p38 MAPK was increased in response to KC-treated
cremaster muscle. The inhibitor SKF86002 had no effect on
phosphorylated p38 levels. The activity of the phosphorylated p38 MAPK,
measured by its ability to phosphorylate ATF-2, was also increased with
KC relative to saline treatment of cremaster muscle. Intravenous
administration of SKF86002 greatly reduced ATF-2 phosphorylation (Fig. 6B). These results are consistent with the inhibitor
blocking p38 MAPK activity. This experiment was repeated three times
with similar results.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
p38 but not the p38
or p38
(21). Not
surprisingly, these inhibitors do not inhibit other MAPK members even
at 100 µM or five times the concentration used in this study
(22). Finally, a great interest has developed in these
inhibitors as they have been shown to have significant therapeutic
benefit in a number of models of inflammation, including endotoxemia
(17, 9), collagen-induced arthritis (17),
pulmonary inflammation (9), and gastritis
(11).
Although p38 MAPK inhibitors significantly reduced neutrophil
recruitment in each of the aforementioned models, the complexity of the
models preempts any possibility of elucidating the mechanism by which
p38 MAPK may contribute to neutrophil recruitment. For example, in
endotoxemia, p38 MAPK inhibitors were shown to reduce TNF production,
which was likely the reason that neutrophil recruitment was reduced
(9). Indeed, in in vitro studies, adhesion molecule
expression has been shown to be reduced and integrin activation is
prevented with p38 MAPK inhibition, potentially reducing the number of
rolling, adhering, emigrating, or migrating (through interstitium)
leukocytes (3, 12, 13). To minimize the biological
complexity, we designed a very simple model that would allow us to
identify each of the steps in the neutrophil recruitment cascade,
including rolling, adhesion, emigration, and directional migration
(chemotaxis) through tissue in response to a single exogenously applied
CXC chemokine. We chose KC, as it is the murine counterpart of human
GRO-, with which it shares 65% sequence identity. KC is a very
potent chemoattractant for neutrophils (23) by activating
the murine IL-8RB homolog, CXCR2 (23). Our data clearly
demonstrate that two p38 MAPK inhibitors, in a dose response, reduced
neutrophil emigration and chemotaxis, but had no effect upon the
upstream events, including rolling and adhesion.
Because the recruitment process is indeed a cascade of events, intervening at one step will inevitably affect downstream events. Therefore, it is possible that the reduction in emigration with the p38 MAPK inhibitors indirectly also attenuated the subsequent chemotaxis through the tissues. However, there are a number of reasons why the reduction in emigration may not account for the reduction in chemotaxis. First, the cells that did emigrate in the presence of p38 MAPK inhibitors failed to chemotax in an effective manner toward the chemokine source. This was best exemplified by the fact that proportionately, a greater inhibition of leukocyte number was seen at increasing distance from the vessel. In a second series of experiments, we only exposed the neutrophils to the p38 MAPK inhibitors after they emigrated out of the vasculature (superfused SKF86002). Using this approach, we were able to see a clear impairment in the ability of the neutrophils to move through the extravascular space. This is an interesting observation in that it suggests that activation of p38 MAPK is an ongoing process that does not require prophylactic treatment. Interruption of p38 MAPK activity stopped the biological endpoint—in this case chemotaxis.
It is tempting to conclude that p38 MAPK has at least two separate
functions: the activation of p38 MAPK is essential for neutrophil
emigration and p38 MAPK contributes to subsequent chemotaxis. However,
the dual function for p38 MAPK may reflect the fact that the neutrophil
uses the same molecular mechanism to migrate between endothelium and
for subsequent chemotaxis and the distinction is simply the two
separate microenvironments (within the microcirculation and outside the
microcirculation). A second possibility is that the two events require
distinct intracellular pathways, with p38 MAPK as an upstream
regulator. Support for the latter is that quite distinct adhesive
mechanisms are used for transendothelial migration and tissue
chemotaxis. PECAM-1 (24), CD47 (25),
and CD18 (26) have been postulated as important adhesion
molecules that contribute to endothelial transmigration. By contrast,
the 1 integrins including
21 have been shown to
be essential for neutrophil chemotaxis in tissues (27). In
fact, it appears that the endothelial transmigration process is
essential for the expression of 1 integrin
molecules (28). Whether p38 MAPK is important in the
1 integrin up-regulation during emigration
remains unclear.
A shortcoming of in vivo experiments is the inability to inhibit p38 MAPK in a single cell type. Therefore, we cannot conclude whether the effect of the p38 MAPK inhibitors is a direct effect upon neutrophils or involves other cells such as endothelial cells. This is not trivial in as much as endothelial cell activation has been shown to be essential for subsequent neutrophil transmigration (29). For example, endothelium cell contraction with subsequent paracellular gap formation is required for neutrophil transendothelial migration in response to chemotactic factors (30). In fact, a very recent in vitro study reported that pretreatment of endothelial cells with SB203580 reduced neutrophil migration on endothelium toward endothelial cell junctions. The impairment in neutrophil migration was a result of inhibiting endothelial cytoskeleton rearrangements (31). Taken together, it is possible that this process is involved in the reduced neutrophil emigration observed in our study. Indeed, we appear to have some indirect evidence that p38 MAPK was activated in parenchymal cells (presumably endothelial cells) because in the complete absence of any neutrophil infiltration in response to KC in anti-P-selectin Ab-treated mice, p38 MAPK activation was still noted (data not shown).
The role of p38 MAPK in neutrophil chemotaxis is supported by the report that installation of KC into lungs induced neutrophil accumulation that was significantly decreased by p38 MAPK inhibition. Although colleagues postulated that this may have been a result of impaired chemotaxis (9), our data extend that work to demonstrate that both endothelial transmigration and tissue chemotaxis were impaired. Interestingly, fMLP but not IL-8 has been shown to activate p38 MAPK and induce neutrophil chemotaxis via this mechanism (6, 32). Although it may seem surprising that KC but not IL8 appears to be dependent upon p38 MAPK, an important difference is that KC appears to stimulate CXCR2 in mice exclusively (23), whereas in humans, IL8 activates both CXCR1 and CXCR2, but the former receptor appears to be more important for chemotaxis (33). Another possible difference is that all of the work with IL8 and p38 MAPK has been completed in circulating cells, whereas our study examined emigrated cells. This is likely a critical difference based on our observation that it is during the emigration process that p38 MAPK becomes important. Clearly, our data indicate that neutrophil locomotion in tissue in response to KC is primarily dependent on p38 MAPK and they suggest a principal role for this intracellular signaling kinase in leukocyte motility.
Chemokines induce morphological changes in leukocytes that include the rearrangement of cytoskeleton, formation of integrin-mediated focal adhesions, and the detachment from the substrate in a coordinated manner with extension and retraction of pseudopods to execute the directional migration (34). Although we have not yet studied the downstream proteins that may be phosphorylated by p38 MAPK that could be involved in neutrophil cytoskeletal rearrangement, a number of candidate molecules should be considered. Molecules associated with motility that are downstream of p38 MAPK include heat shock protein 27. This molecule is involved in the regulation of actin assembly and has been shown to be important in smooth muscle cell contraction (35). In addition, leukocyte-specific gene 1 (LSP1) protein is an F-actin binding protein and a major substrate of MAPK-activated protein kinase 2 in neutrophils (36). In vitro work has shown that neutrophils from LSP1 knockout mice showed impaired chemotaxis in response to KC (37). Taken together with our data, we would propose that KC activates p38 MAPK, which phosphorylates downstream proteins like LSP-1 to permit neutrophil migration through tissue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Kubes, Health Sciences Center, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada. E-mail address: pkubes{at}ucalgary.ca
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; KC, keratinocyte-derived cytokine; LSP1, leukocyte-specific gene 1.
Received for publication July 11, 2001. Accepted for publication September 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or formyl-methionyl-leucyl-phenylalanine stimulation. J. Immunol. 160:1982.-induced E-selectin expression is activated by the nuclear factor-
B and c-JUN N-terminal kinase/p38 mitogen-activated protein kinase pathways. J. Biol. Chem. 272:2753.2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst. J. Immunol. 161:1921.4-integrin, P-selectin, and E-selectin in an allergic model of inflammation. J. Exp. Med. 185:1077., , , and in inflammatory cell lineages. J. Immunol. 162:4246.21 (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue. Blood 95:1804.1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration. FASEB J. 9:1103.[Abstract]
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M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
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S. Vitale, A. Schmid-Alliana, V. Breuil, M. Pomeranz, M.-A. Millet, B. Rossi, and H. Schmid-Antomarchi Soluble Fractalkine Prevents Monocyte Chemoattractant Protein-1-Induced Monocyte Migration via Inhibition of Stress-Activated Protein Kinase 2/p38 and Matrix Metalloproteinase Activities J. Immunol., January 1, 2004; 172(1): 585 - 592. [Abstract] [Full Text] [PDF]
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J. Gomez-Cambronero, J. Horn, C. C. Paul, and M. A. Baumann Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway J. Immunol., December 15, 2003; 171(12): 6846 - 6855. [Abstract] [Full Text] [PDF]
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 C. E. Green, D. N. Pearson, N. B. Christensen, and S. I. Simon Topographic requirements and dynamics of signaling via L-selectin on neutrophils Am J Physiol Cell Physiol, March 1, 2003; 284(3): C705 - C717. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, P. G. Arndt, J. G. Lieber, B. T. Suratt, K. R. Poch, N. J. Avdi, K. C. Malcolm, C. Taube, P. M. Henson, et al. Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase J. Immunol., November 1, 2002; 169(9): 5260 - 5269. [Abstract] [Full Text] [PDF]
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 B. Heit, S. Tavener, E. Raharjo, and P. Kubes An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients J. Cell Biol., October 14, 2002; 159(1): 91 - 102. [Abstract] [Full Text] [PDF]
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D.-M. McCafferty, A. W. B. Craig, Y. A. Senis, and P. A. Greer Absence of Fer Protein-Tyrosine Kinase Exacerbates Leukocyte Recruitment in Response to Endotoxin J. Immunol., May 15, 2002; 168(10): 4930 - 4935. [Abstract] [Full Text] [PDF]
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