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Department of Surgery, Toronto Hospital and University of Toronto, Toronto, Ontario, 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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Recent studies have suggested that hyperosmolar solutions exert alterations in immune cell function that may contribute to improved outcome (7, 8, 9). Junger and colleagues reported the ability of HTS resuscitation to reverse hemorrhage-induced suppression of splenocyte proliferation in a murine model (8). In subsequent experiments, these investigators demonstrated that hyperosmolarity can enhance mitogen-stimulated T cell proliferation in vitro through a mechanism involving tyrosine phosphorylation and activation of the p38 mitogen-activated protein kinase. The reversal of trauma-induced immunosuppression by HTS may represent a mechanism by which this resuscitation strategy may lessen septic complications and thus enhance survival.
Significant morbidity in the trauma patient also results from the development of multiple organ dysfunction, even in the absence of infection (10). This process is predominantly mediated by the sequestration of activated neutrophils within the microvasculature and subsequent release of their injurious products, such as reactive oxygen species and proteolytic enzymes. The lung appears to be a particular target, with the appearance of the acute respiratory distress syndrome in up to 40% of the traumatized patient population (10, 11). Hemorrhagic shock is believed to contribute to the pathogenesis of acute respiratory distress syndrome by rendering the patient more susceptible to a second, seemingly trivial, inflammatory stimulus, the so-called two-hit model (12, 13). In this regard, previous studies have suggested that the process of ischemia-reperfusion might mediate this effect by priming circulating neutrophils for increased superoxide production, and therefore enhanced cytotoxicity, once they are sequestered in the lung (12, 13).
Previous reports have documented the ability of hyperosmolar solutions to inhibit a variety of neutrophil functions, including the generation of reactive oxygen species (14, 15). We therefore hypothesized that HTS resuscitation from hemorrhagic shock might lessen the development of subsequent lung injury. To test this possibility, we used a rodent model in which resuscitated hemorrhagic shock primes for increased pulmonary leukosequestration and albumin leak in response to a small dose of intratracheal LPS (16). The data demonstrate that HTS resuscitation from shock markedly reduced lung injury in response to LPS by preventing neutrophil sequestration. This appeared to be predominantly mediated via an effect on neutrophil adhesion molecule expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ficoll and dextran T500 were purchased from Pharmacia Biotech
(Baie D’Urfe, Canada). Trizma (Tris base), 2-ME, FMLP, BSA, PMA,
citrate, LPS (Escherichia coli O111: B4), propidium
iodide, sarcosyl, and guanidine isothiocyanate were obtained from Sigma
(St. Louis, MO). [125I]albumin was purchased from Frosst
(Kirkland, Canada), EDTA from BDH (Toronto, Canada), Triton X-100 from
Caledon (Georgetown, Canada), and 51Cr from Amersham
(Oakville, Canada). Cell Wells tissue culture chambers were from
Corning Costar (Cambridge, MA). Glyceraldehyde-3-phosphate
dehydrogenase G3PDH was obtained from Clontech (Palo Alto, CA), gelatin
from Bio-Rad (Hercules, CA), diS-C3- (5) from Molecular Probes (Eugene,
OR), and human fibronectin from Boehringer Mannheim (Laval, Canada).
Human recombinant TNF-
, valinomycin, and gramicidin were purchased
from Calbiochem (La Jolla, CA). The anesthetic drugs used were
pentobarbital from MTC Pharmaceuticals (Cambridge, Canada), ketamine
from Rogar/STB (London, Canada), and xylazine from Bayer (Etobicoke,
Canada). The rat neutrophil isolating kit NIM2 was purchased from
Cardinal Associates (Santa Fe, NM). All chemicals used were of the
highest purity available.
Solutions
DMEM, HBSS, medium 199, and PBS were obtained from Life Technologies (Burlington, Canada). Ringer’s lactate (RL) was obtained from Baxter (Toronto, Canada) ,and 10% buffered formalin was purchased from Fisher Chemical (Fairlawn, NJ). PMNs were isolated in DMEM containing 10% FCS (HyClone, Logan, UT) and penicillin/streptomycin (Life Technologies). TBS contained 100 mM NaCl and 50 mM Tris, pH 7.4. Isotonic NaCl solution contained 3 mM KCl, 5 mM glucose, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES, pH 7.4. Hypertonic medium was obtained by adding extra NaCl to the DMEM containing 10% FCS. The Toronto Hospital Pharmacy Department prepared HTS/7.5% NaCl for use in resuscitation of animals.
Abs and cell line
All Abs were monoclonal. The following were anti-human: FITC-labeled anti L-selectin Dreg 56 from Immunotech (Marseille, France) and anti-CD11b (Leu 15)-phycoerythrin from Becton Dickinson (San Jose, CA). The following were anti-rat: FITC-labeled anti-L-selectin HRL2 from Sekagaku (Tokyo, Japan), and FITC-labeled anti-CD11b OX-42 and anti-ICAM-1 (both from Serotec, Oxford, U.K.). ECV304 (HUVEC line) was obtained from American Type Culture Collection (Manassas, VA).
Animal model
Adult male Sprague-Dawley rats, weighing 300–350 g (Charles River, St. Constant, Canada), were anesthetized with i.p. ketamine (80 mg/kg) and xylazine (8 mg/kg). The right carotid artery was cannulated with a 22-gauge angiocath (Becton Dickinson, Franklin Lakes, NJ) for monitoring of mean arterial pressure (MAP), blood sampling, and resuscitation. Hemorrhagic shock was initiated by blood withdrawal and reduction of the MAP to 40 mm Hg within 15 min. This blood pressure was maintained by further blood withdrawal if the MAP was >45 mm Hg, and by infusion of 0.5 ml of RL if the MAP was <35 mm Hg. Shed blood was collected into 0.1 ml of citrate/ml blood to prevent clotting. After a hypotensive period of 60 min, animals were resuscitated by transfusion of the shed blood and either an equivalent amount of RL or a 4 ml/kg volume of 7.5% NaCl (HTS) over a period of 2 h. The catheter was then removed, the carotid artery was ligated, and the cervical incision was closed.
One hour later, 100 µg of LPS in 200 µl of saline was administered intratracheally through a 14-gauge angiocath tracheotomy. Sham animals were instrumented but not bled, and saline alone was instilled intratracheally. Animals were sacrificed by pentobarbital overdose at various time points depending on the specific study. Blood samples were collected during the experiment for serum osmolarity. Serum was separated by centrifugation, and osmolarity was measured in an automated osmometer Advanced Osmometer 3D3 (Two Technology Way, Norwood, MA). For histologic assessment, the whole lungs were fixed in 10% neutral buffered formalin (pH 7.4) and later stained with hematoxylin/eosin and examined using an optical microscope.
Bronchoalveolar lavage (BAL)
BAL cell counts were determined at 4 h following LPS or saline intratracheal instillation. Immediately after sacrifice, the lungs were perfused via the tracheotomy cannula with cold PBS/0.1 mM EDTA in 10-ml aliquots and were gently withdrawn to a total of 40 ml. For cell counts and differential, BAL fluid was centrifuged at 300 x g for 10 min. After discarding the supernatant, the pelleted cells were resuspended in serum-free DMEM. Total cell counts were made on a grid hemocytometer, and the differential cell counts were made on a cytospin-prepared slide stained with Wright-Giemsa. A total of 500 cells were counted in cross-section per sample, and the number of neutrophils was calculated as follows: number of neutrophils = total cell count x % of neutrophils in BAL fluid sample.
Myeloperoxidase (MPO) assay
Lung samples were thawed, and approximately 0.5 g of tissue was homogenized in 25 ml of potassium phosphate (10 mM; pH 7.4) for 1 min using a Brinkmann Polytron (model PT10/35, Brinkmann Instruments, Westbury NY). The homogenate was centrifuged at 12,000 x g for 20 min at 4°C. The supernatant was discarded, and the pellet was resuspended and homogenized in 25 ml of potassium phosphate (50 mM; pH 6.0) containing 0.5% HTAB. The homogenate was frozen overnight at -70°C, rehomogenized for 1 min, and sonicated (model VC 50T, Sonics & Materials, Danbury, CT) at 40 W for 1 min. After centrifugation as described above, the supernatant was collected and used for both MPO and protein assays.
MPO activity was assessed by colorimetric assay using H2O2 as a substrate as previously described (17). The absorbance change per minute was used as a measure of MPO activity. Results are expressed as MPO activity per milligram of protein. Protein concentrations were determined using the Pierce bicinchoninic acid protein assay (Rockford, IL).
Transpulmonary albumin flux
Rats were injected with 1 µCi of [125I]albumin into the tail vein immediately preceding intratracheal LPS or saline. Six hours later, both 1 ml of blood collected by cardiac puncture and three samples of 1 ml of BAL fluid were analyzed by a gamma spectrometer. The [125I]albumin leak was corrected to blood counts per minute and expressed as the transpulmonary albumin leak index as follows: transpulmonary albumin leak index = (cpm/ml in BAL fluid)/(blood cpm/ml).
Neutrophil isolation and labeling
Human neutrophils. Cells were isolated from fresh blood drawn by venipuncture into heparinized tubes. Isolation was performed using 3% dextran sedimentation and centrifugation on Ficoll-Hypaque cushions as described previously (18). After erythrocyte lysis in 0.84% NH4Cl, the PMNs were resuspended at a cell density of 1 x 106 PMN/ml in DMEM and were used immediately after isolation. Neutrophil viability exceeded 97% as assessed by trypan blue exclusion.
Rat neutrophils. Isolation was performed according to the NIM2 manufacturer’s instructions (Cardinal Associates, Santa Fe, NM). Briefly, after sacrifice, the total blood volume of the animal was obtained by cardiac puncture and collected in a citrated glass tube. The blood was layered onto two discontinuous Ficoll-Hypaque gradients (NIM2). After centrifugation at room temperature (400 x g for 45 min) the pelleted cells were washed in HBSS once, and the erythrocytes were lysed in erythrocyte lysing buffer (0.84% NH4Cl). Finally, the cells were resuspended in DMEM. Isolated cells consisted of >90% PMN with >95% viability by trypan blue dye exclusion.
Neutrophil labeling with 51Cr. For labeling rat neutrophils, 5 x 106 PMN were resuspended in 0.4 ml of DMEM and incubated for 20 min with 40 µCi of 51Cr at 38°C. Following the labeling period, cells were washed twice and incubated in either isotonic medium or hypertonic medium (350 mosmol) for 4 h. In some studies, freshly isolated cells were used immediately following the labeling step. Cells were pelleted by centrifugation at 300 x g and were resuspended in isotonic DMEM. An aliquot of cells from each group was counted to ensure equivalent labeling (1480 Wizard 3 automatic gamma counter, Wallac, Turku, Finland). There was no difference in the labeling between isotonic and hypertonic treatments.
Detection of radiolabeled PMN in the lung
In some studies, we examined whether isolated rat neutrophils exposed to isotonic or hypertonic medium ex vivo might exhibit differential sequestration in the lung. Rats underwent either sham manipulation or hemorrhagic shock with RL resuscitation as described. Following PMN isolation, ex vivo labeling, and incubation in isotonic or hypertonic solution as described above, 2 x 10651Cr-labeled isotonic medium or hypertonic medium, resuspended in isotonic medium, was injected into the tail vein of the rats immediately before intratracheal LPS or saline instillation. At sacrifice 4 h later, 1 ml of blood was collected by cardiac puncture. The lungs were harvested after being flushed with PBS/EDTA through a cannula inserted in the pulmonary veins until the effluent was completely clear. 51Cr-labeled PMN accumulation in the lung was analyzed in the gamma spectrometer and calculated as follows: 51Cr-labeled PMN lung accumulation = (lung cpm/g tissue)/(blood cpm/ml).
Assessment of neutrophil viability
At the end of the preparation phase, two approaches were used to assess PMN viability. By trypan blue exclusion technique, PMN viability exceeded 95% and did not differ between isotonic and hypertonic treatment. Measurement of transmembrane potential was also tested using the anionic fluorescent probe diS-C3 (5) as previously described (19).
Measurement of neutrophil surface CD11b and L-selectin
In vitro studies on human PMN. Flow cytometric analysis was used to evaluate surface expression of these adhesion molecules. Isolated human PMN were incubated in isotonic medium or hypertonic medium at 350 or 500 mosmol for up to 4 h. PMN were resuspended in isotonic medium and incubated with FITC-labeled anti-L-selectin or anti-CD11b Ab at a 1/10 dilution for 20 min at 4°C. PMNs were then analyzed on the FACScan (Becton Dickinson, Palo Alto, CA) using an FL1 detector (488 nm excitation wavelengths). Typically, 5,000 cells were analyzed per condition, and the results were expressed as the mean channel fluorescence. CD11b expression was measured in resting and stimulated human PMN. The following stimuli were used: LPS (1 µg/ml for 1 h), PMA (50 nM for 20 min), or FMLP (10-7 M for 20 min).
Detection of surface markers on rat PMN in whole blood. For CD11b detection, 100 µl of whole rat blood was incubated with FITC-labeled anti-CD11b Ab at 1/10 dilution at room temperature for 20 min. Erythrocytes were lysed with lysing buffer (0.84% NH4Cl), the remaining cells washed twice with DMEM and analyzed on the FACScan as above. For measurement of L-selectin, PMNs were isolated from whole blood and incubated with FITC-labeled L-selectin at a 1/10 dilution for 20 min at 4°C. PMNs were then analyzed on the FACScan as described above.
Neutrophil adhesion assays
ECV304 human endothelial cells were grown to confluence on
fibronectin-coated tissue culture chambers. To induce high expression
of ICAM-1 (25) the ECV304 cells were stimulated with TNF- (1 ng/ml)
for 6 h, and subsequently washed with fresh DMEM and 10% FCS.
Neutrophils were treated with iso- or hypertonic (350 mosmol) medium
for 4 h, then incubated in the presence or the absence of LPS (1
µg/ml) for 1 h. Next, all neutrophils were resuspended in
isotonic DMEM and 10% FCS and layered on top of the prestimulated
endothelial cells for 30 min at 37°C. Nonadherent neutrophils were
removed by repeated washes with PBS. The number of adherent neutrophil
was determined by microscopy by two investigators blinded to the
manipulations. Three high-power fields were counted per well, and each
group was plated in six different wells.
Northern blot analysis
Total RNA from lungs was obtained using the guanidium-isothiocyanate method (20). Briefly, lungs were harvested and homogenized in 10 ml of 4 M guanidine isothiocyanate containing 25 mM sodium citrate, 0.5% sarcosyl, and 100 mM ß-ME. RNA was denatured, electrophoresed through a 1.2% formaldehyde-agarose gel, and transferred to nylon membrane. Hybridization was conducted using a 32P-labeled random-primed murine ICAM-1 cDNA probe (American Type Culture Collection) and a [32P]dATP end-labeled 30-base oligonucleotide probe for cytokine-induced neutrophil chemoattractant (CINC) with the sequence 5'-GCGGCATCACCTTCAAACTCTGGATGTTCT-3', which is complimentary to nucleotides 134–164 of CINC cDNA (21) provided by Dr. Timothy S. Blackwell (Vanderbilt University School of Medicine, Nashville, TN). Blots were then washed under conditions of high stringency, and specific mRNA bands were detected by autoradiography in the presence of intensifying screens as previously reported (22). To control for loading, blots were then stripped and reprobed for G3PDH, a ubiquitously expressed housekeeping gene (23). Expression of mRNA was quantitated using a PhosphorImager and accompanying ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and was normalized to the G3PDH signal.
Western blot analysis
To study ICAM-1 protein expression in the lung, whole rat lungs were homogenized in TBS/1% Triton X-100 solution. Lung tissue homogenate samples were separated on a 15% SDS-PAGE under nonreducing conditions (24). Separated proteins were electroblotted onto polyvinylidene difluoride membrane and blocked for 1 h at room temperature with Tris-buffered saline containing 1% BSA. The membranes were then incubated with a 1/1000 dilution of antiserum against ICAM-1 at room temperature for 1 h. Ag-Ab complexes were identified with goat anti-rabbit IgG tagged with horseradish peroxidase (Sigma, St. Louis, MO) and detected using the enhanced chemiluminesence detection system (Amersham, Arlington Heights, IL).
Statistical analysis
Data are presented as the mean ± SE of the number of experiments indicated. When blots are shown, they are representative of at least three independent studies. Significance was assessed using Student’s t test and one-way analysis of variance with post-hoc testing with the Tukey-Kramer multiple comparisons test. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal survival at the end of the experimental period in both
resuscitation groups exceeded 90%, with deaths due to tracheal
obstruction secondary to the presence of the intratracheal cannula. HTS
resuscitation effectively restored MAP to preshock levels (data not
shown). Fig. 1
illustrates the serum
osmolarity at various time points following resuscitation with HTS or
RL. As shown, HTS caused a rapid increase in osmolarity that peaked at
approximately 500 mosmol at 5 min and then returned to approximately
350 mosmol by 1 h. This was sustained over the ensuing 3 h.
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Our laboratory has previously reported that resuscitated shock
primes the lung for increased tranpulmonary albumin leak in response to
LPS (16). As demonstrated in Fig. 2, both
shock alone and LPS alone caused small increases in albumin leak. The
administration of LPS following shock resuscitated with RL caused a
further significant rise in the albumin leak. However, when shocked
animals were resuscitated with HTS, the increase in albumin leak was
totally prevented.
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A shows alveolar PMN counts
under each of the conditions. Compared with shock/LPS animals
resuscitated with RL, the use of HTS caused >50% reduction in the
number of PMN recovered in the BAL. As a measure of overall lung
sequestration of PMNs, lung MPO levels were studied. As demonstrated in
Fig. 3B, HTS resuscitation caused reduced levels of MPO
compared with those in animals resuscitated with RL. Reduced lung
inflammation with HTS resuscitation was also seen in the histopathology
of the lung (Fig. 4). Shock animals
resuscitated with RL before LPS administration (left
panel) exhibited neutrophilia in the interstitium and in the
alveolar space, while shock/LPS animals repleted with HTS demonstrated
lung histology comparable to that of sham animals, with few neutrophils
and minimal alveolar hemorrhage and edema. Considered together, these
data suggest that the protective effect of HTS resuscitation may be
related to the reduced pulmonary leukosequestration.
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We recently showed that augmented pulmonary PMN sequestration in
this model was mediated by increased expression of the C-X-C chemokine
CINC in the lung (16). To determine whether HTS might exert its effect
by suppressing CINC expression, we studied CINC mRNA in whole lungs of
animals resuscitated with RL or HTS. As shown in Fig. 5, the shock/LPS-induced increase in CINC
mRNA did not differ between RL and HTS infusion. A representative blot
is shown in Fig. 5A, and the mean of multiple studies
normalized for G3PDH is shown in Fig. 5B. These data suggest
that altered CINC does not account for the attenuation of PMN
accumulation following HTS.
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To discern whether the changes in PMN function were sufficient to
account for the reduced pulmonary leukosequestration, PMNs were labeled
ex vivo, exposed to isotonic or hypertonic medium for 4 h, and
then resuspended in isotonic medium. They were then reinfused into sham
animals or animals following RL resuscitation from shock, immediately
before LPS administration (Fig. 6A). As expected, sham animals
given isotonically treated PMN or hypertonically treated PMN did not
exhibit PMN accumulation in the lung. Further, shocked animals
resuscitated with RL showed increased PMN counts in the lung when
isotonic medium-treated PMN were given. However, PMN exposed to
hypertonicity ex vivo failed to sequester in the lung. The viability of
cells following in vitro treatment was confirmed by their capacity to
exclude trypan blue. The integrity of the cell membrane (i.e., low
permeability to monovalent ions) was also demonstrated by the fact that
hypertonically treated cells maintained their transmembrane potential
and were able to hyperpolarize after the addition of the potassium
ionophore valinomycin (Fig. 6B). Considered together, these
data support the concept that hypertonicity may exert its protection by
a direct effect on PMNs, which precludes their sequestration in the
lung.
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The initial steps leading to PMN sequestration in
the vasculature involve interactions between neutrophil surface
receptors and counterligands on endothelial cells (25). To evaluate
whether HTS might impair PMN adhesion molecule expression and thus
contribute to the reduced PMN sequestration, blood was obtained from
animals following each resuscitation protocol and was evaluated for
adhesion molecule expression. Fig. 7
demonstrates CD11b expression in vivo in animals resuscitated with RL
or HTS. RL animals showed a significant increase in CD11b at the end of
resuscitation compared with the preshock value. This was sustained over
the ensuing 4 h until the time of sacrifice. By contrast, in the
HTS-treated animals, CD11b levels did not increase compared with the
preshock value over this time period. Fig. 8 shows levels of L-selectin in blood
PMNs following the two resuscitation regimens. In animals resuscitated
with RL, L-selectin levels remained stable over time, while in
HTS-treated animals, there was a significant loss of surface L-selectin
expression.
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A). CD11b expression on
freshly isolated cells stimulated with FMLP or LPS did not differ from
the expression observed after a 4-h incubation in isotonic medium. The
level of CD11b expression on PMA-stimulated freshly isolated cells was
approximately 35% higher than that following a 4-h incubation in
isotonic medium (Fig. 9A). Several studies have suggested
that increased CD11b expression is not necessary for augmented function
(26, 27). To discern whether exposure of neutrophils to hypertonicity
alters CD11b function, adhesion to endothelial cells was evaluated
(Fig. 9B). As expected, LPS-stimulated neutrophils exhibited
increased adhesion to an endothelial cell monolayer pretreated with
TNF-. However, this rise was inhibited by pretreatment with
hypertonic medium. Considered together, these findings suggest that
hypertonicity is able to impair both LPS-induced up-regulation of CD11b
surface expression and its activation. In addition, incubation in 350
mosmol in vitro caused a time-dependent reduction in L-selectin levels,
an effect that was augmented by increasing the osmolarity to 500 mosmol
(Fig. 9C). L-selectin levels on cells incubated for 4 h
in isotonic medium did not differ from those in fresh cells and were
significantly higher than those in cells incubated for 4 h in
either 350 or 500 mosmol (Fig. 9C).
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Endothelial cell ICAM-1 is an important ligand for neutrophil
CD11b and has been implicated in mediating neutrophil-endothelial
interaction at sites of inflammation (25, 28). While HTS-induced
alterations in PMNs were shown to be sufficient to account for the
reduced leukosequestration, this did not rule out the possibility that
endothelial cells might themselves be affected by this regimen. To
determine the effect of HTS resuscitation on ICAM-1 expression, we
examined ICAM-1 mRNA and protein expression in the lungs of animals
following shock/LPS and either RL or HTS resuscitation. Four hours
after LPS infusion, the RL-resuscitated animals showed a marked
increase in ICAM-1 mRNA expression, as shown in a representative blot
(Fig. 10A). Densitometric
evaluation of several studies revealed an approximately 50% reduction
in ICAM-1 mRNA expression in animals resuscitated with HTS (Fig. 10B). Similar reductions in whole lung ICAM-1 protein were
observed in HTS-resuscitated animals compared with RL-treated animals
(Fig. 10C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMNs transiently incubated in hyperosmolar solution ex vivo failed to sequester in lungs of shock/LPS animals resuscitated with RL. This finding suggests that hypertonicity-induced changes in circulating PMNs may be sufficient to account for the salutary effect of HTS resuscitation in this model. In this regard, blockade of surface integrins or selectin molecules has been shown to prevent neutrophil accumulation in various models of inflammation (25, 29, 30, 31). In the present studies, both in vivo and in vitro data suggest that hypertonicity might exerts its effect on PMNs through altering adhesion molecule expression: L-selectin via increased shedding and CD11b through impaired LPS-stimulated up-regulation and activation. The mechanisms underlying these alterations warrant further investigation. In principle, both the induction of volume-sensitive signaling cascades as well as shrinkage-related changes in the cytoskeleton might contribute to the overall effect. For example, osmotic shrinkage has been reported to cause extensive tyrosine phosphorylation in neutrophils and fibroblasts (18, 32). This may be caused by clustering of surface receptors related to osmotic shrinkage, a process that has been shown to stimulate various receptors on the surface of HeLa in the absence of their ligands (33). Relevant to the present studies, clustering of the Leu 13 receptor on lymphocytes has been shown to cause L-selectin shedding through a tyrosine kinase-dependent mechanism (34). While cellular activation might be expected to lead to concomitant up-regulation of CD11b and activation, hypertonicity alone did not alter surface CD11b expression and actually inhibited its stimulated up-regulation in vivo. In vitro, both the level of CD11b expression and its activation in response to LPS were impaired by hypertonicity. Using an in vitro system, Hampton and colleagues reported that hyperosmolarity in the range of 500–700 mosmol significantly impaired FMLP-stimulated degranulation of secondary granules in neutrophils, as evidenced by reduced release of the granular enzyme lysozyme (15). A comparable mechanism for impaired LPS-induced up-regulation of CD11b might be invoked, given that CD11b is also present to some extent in secondary granules as well as in secretory vesicles in these cells (35). Recent studies testing the effect of the actin-polymerizing agent jasplakinolide on neutrophil function revealed that this agent was able to prevent FMLP-induced expression of CD11b in neutrophils (36), suggesting that the state of the actin cytoskeleton might influence exocytic translocation of receptors to the cell surface. The cellular filamentous actin content is known to be increased following hypertonicity-induced cell shrinkage (37) and may therefore have similarly contributed to the impaired LPS-stimulated increase in CD11b both in vivo and in vitro.
In the present studies the investigations regarding altered neutrophil sequestration focused on the effect of hypertonicity on the expression of CD11/CD18 and L-selectin. The rationale for choosing these particular adhesion molecules was based on previous studies demonstrating their role in lung neutrophil accumulation and subsequent tissue damage in models of acute lung injury. For example, inhibitory anti-CD18 mAb was shown to markedly reduce neutrophil emigration into the alveolar space of rabbits following intratracheal instillation of LPS (31). Similarly, anti-L-selectin Abs diminished lung neutrophil accumulation in a rodent ischemia/reperfusion model as well as in an injury model induced by intrapulmonary instillation of IgG complexes (38, 39). However, particularly in the first model, inhibition of leukocyte accumulation was incomplete, suggesting a role for CD18-independent mechanisms. Recent studies using adhesion molecule-deficient mice have provided significant support for this concept. In both L-selectin-deficient (40, 41) as well as CD18-deficient mice (41), neutrophil emigration into the lung in response to intratracheal challenge with either live Escherichia coli or Streptococcus pneumoniae was actually increased compared with that in wild-type animals. Whether intratracheal LPS in these animals will result in comparable findings of CD18- and L-selectin-independent neutrophil sequestration or findings similar to those reported in the aforementioned Ab studies remain to be tested. However, since the requirement for a particular adhesion molecule in pulmonary leukosequestration is markedly dependent on the inflammatory stimulus (31, 39), previous work would suggest that in the shock/LPS model used in the present studies, a prominent role for CD11/CD18 might be predicted. Based on this consideration, it seems plausible that the ability of hypertonic medium to inhibit up-regulation and activation of this adhesion complex might contribute significantly to the inhibition of neutrophil sequestration following hypertonic resuscitation. The approximately 30% reduction in L-selectin levels in hypertonic resuscitated animals may be contributory, although alone it is unlikely to have been responsible for the observed protection. Other adhesion molecules may also be important and require further study.
During the process of transmigration, neutrophils have been shown to swell by up to 60% (42, 43). Preventing this volume increase in vitro using hyperosmolar solutions (350 mosmol) inhibited the chemotactic index by approximately 50% (43). In the present studies we did not specifically evaluate the relative effects of hypertonicity on leukocyte sequestration in the lung vasculature vs transmigration of neutrophils into the alveolar space. However, two lines of evidence suggest that reduced sequestration may have accounted for the effect. First, lung MPO levels, a measure of overall PMN sequestration, were lowered concomitant with the reduction in BAL neutrophils. Second, PMNs exposed to a hypertonic incubation ex vivo failed to accumulate in the lung tissue. While these data together implicate reduced sequestration as a mechanism for the effect of HTS, we cannot rule out the possibility that reduced transmigration may have additionally contributed to the diminution of BAL neutrophil counts in HTS-resuscitated animals.
Angle and colleagues recently reported that HTS resuscitation in a mouse hemorrhagic shock model caused a modest reduction in the lung injury score as well as an improvement in the histopathologic appearance of the lung (9). Neutrophil CD11b levels did not differ between control and HTS-resuscitated animals, while L-selectin, chemokine, and endothelial cell adhesion molecules were not examined. Importantly, the murine model differs significantly from the two-hit model in the rat presented herein, in that hemorrhage/resuscitation alone in the mouse suffices to induce inflammation in the lung concomitant with increased expression of proinflammatory and immunoregulatory cytokines (9). In man, hemorrhage/resuscitation alone rarely initiates such a profound response, suggesting that the two-hit model may be more representative of human disease (12, 13). Nevertheless, the murine model may prove valuable in dissecting out some of the mechanistic aspects of the salutary effects of hypertonic resuscitation.
In summary, the present studies demonstrate a beneficial immunomodulatory effect of hypertonic resuscitation on the development of lung injury. This effect appears to be due to an inability of circulating neutrophils to sequester at the site of injury. These findings suggest that the induction of a transient serum hyperosmolarity might minimize tissue injury in other inflammatory processes that are characterized by neutrophil infiltration and damage, such as those that occur following ischemia/reperfusion.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ori D. Rotstein, Toronto Hospital-General Division, 200 Elizabeth St., EN9–232, Toronto, Ontario, Canada M5G 2C4. E-mail address:
3 Abbreviations used in this paper: HTS, hypertonic saline; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; RL, Ringer’s lactate; MAP, mean arterial pressure; BAL, bronchoalveolar lavage; MPO, myeloperoxidase; CINC, cytokine-induced neutrophil chemoattractant.
Received for publication April 30, 1998. Accepted for publication July 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and neutrophilic lung inflammation. J. Immunol. 157:1630.[Abstract]
mß2) in human neutrophils. J. Clin. Invest. 92:1467.
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 A. Vercueil, D. Levett, and M. Grocott Resuscitation fluids in trauma, part II: which fluid should I give? Trauma, April 1, 2006; 8(2): 111 - 121. [PDF]
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 Y. Chen, N. Hashiguchi, L. Yip, and W. G. Junger Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1051 - C1059. [Abstract] [Full Text] [PDF]
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 M. M. Yada-Langui, E. A. Anjos-Valotta, P. Sannomiya, M. Rocha e Silva, and R. Coimbra Resuscitation Affects Microcirculatory Polymorphonuclear Leukocyte Behavior After Hemorrhagic Shock: Role of Hypertonic Saline and Pentoxifylline Experimental Biology and Medicine, July 1, 2004; 229(7): 684 - 693. [Abstract] [Full Text] [PDF]
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 Y. Chen, A. Shukla, S. Namiki, P. A. Insel, and W. G. Junger A putative osmoreceptor system that controls neutrophil function through the release of ATP, its conversion to adenosine, and activation of A2 adenosine and P2 receptors J. Leukoc. Biol., July 1, 2004; 76(1): 245 - 253. [Abstract] [Full Text] [PDF]
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T. Orlic, W. H. Loomis, A. Shreve, S. Namiki, and W. G. Junger Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1261 - C1269. [Abstract] [Full Text] [PDF]
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 J. Cuschieri, D. Gourlay, I. Garcia, S. Jelacic, and R. V. Maier Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin J. Immunol., February 1, 2002; 168(3): 1389 - 1396. [Abstract] [Full Text] [PDF]
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 D. Cataldo, J.-M. Foidart, L. Lau, P. Bartsch, R. Djukanovic, and R. Louis Induced Sputum : Comparison Between Isotonic and Hypertonic Saline Solution Inhalation in Patients With Asthma Chest, December 1, 2001; 120(6): 1815 - 1821. [Abstract] [Full Text] [PDF]
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 Y. Song, L. Ao, C. D. Raeburn, C. M. Calkins, E. Abraham, A. H. Harken, and X. Meng A low level of TNF-{alpha} mediates hemorrhage-induced acute lung injury via p55 TNF receptor Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L677 - L684. [Abstract] [Full Text] [PDF]
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M. Thiel, F. Buessecker, K. Eberhardt, A. Chouker, F. Setzer, U. Kreimeier, K.-E. Arfors, K. Peter, and K. Messmer Effects of hypertonic saline on expression of human polymorphonuclear leukocyte adhesion molecules J. Leukoc. Biol., August 1, 2001; 70(2): 261 - 273. [Abstract] [Full Text] [PDF]
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S. B. Rizoli, O. D. Rotstein, J. Parodo, M. J. Phillips, and A. Kapus Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling Am J Physiol Cell Physiol, September 1, 2000; 279(3): C619 - C633. [Abstract] [Full Text] [PDF]
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 J. Fan, A. Kapus, Y. H. Li, S. Rizoli, J. C. Marshall, and O. D. Rotstein Priming for Enhanced Alveolar Fibrin Deposition after Hemorrhagic Shock . Role of Tumor Necrosis Factor Am. J. Respir. Cell Mol. Biol., April 1, 2000; 22(4): 412 - 421. [Abstract] [Full Text]
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 S. B. Rizoli, O. D. Rotstein, and A. Kapus Cell Volume-dependent Regulation of L-selectin Shedding in Neutrophils. A ROLE FOR p38 MITOGEN-ACTIVATED PROTEIN KINASE J. Biol. Chem., July 30, 1999; 274(31): 22072 - 22080. [Abstract] [Full Text] [PDF]
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A. Lewis, C. Di Ciano, O. D. Rotstein, and A. Kapus Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization Am J Physiol Cell Physiol, February 1, 2002; 282(2): C271 - C279. [Abstract] [Full Text] [PDF]
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