|
|
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ARTICLE |
CORRESPONDENCE Beverly H. Koller: treawouns{at}aol.com
 Top ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Bacterial pneumonia remains a leading cause of death in humans, especially when complicated with bacteremia and sepsis. Conversely, sepsis resulting from primary infection at other sites is often complicated by the development of acute respiratory distress syndrome, and in fact, sepsis accounts for as many as half of all cases of acute respiratory distress syndrome (1–3). Lung injury often occurs at the onset of bacteremia, resulting in the failure of this organ. In both bacterial pneumonia and acute lung injury secondary to bacteremia, pathophysiological changes include compromise of the alveolar-capillary barrier which results in edema and consequently intravascular shunting and poor ventilation of the alveolus. Studies of sterile acute respiratory distress in dogs over 15 years ago suggested that the loop diuretic furosemide could decrease the intravascular shunt and improve pulmonary oxygen exchange (4). Clinical studies also suggested that this loop diuretic could improve pulmonary function of premature infants with chronic lung disease (5).
Loop diuretics such as furosemide and bumetanide block the activity of the two isoforms of the Na+-K+-2Cl– transporter, NKCC1 and NKCC2. Na+-K+-2Cl– cotransporters couple the electroneutral cellular uptake of one molecule of both Na+ and K+ with two molecules of Cl– across the cell membrane. Under normal physiological conditions, NKCC cotransporters mediate a net influx of ions into cells because of the inwardly directed chemical gradients for Na+ and Cl– ions (6, 7). These systems are present in a broad spectrum of eukaryotic cells and are believed to play a central role in vectorial salt transport across epithelia and volume regulation in epithelial and nonepithelial cells (8, 9). Molecular studies have identified two isoforms, NKCC1 and NKCC2, that share
The expression pattern of the NKCC1 gene (Slc12a2) differs markedly from that of NKCC2 (16–18). NKCC1 is also expressed at high levels in the kidney, where in contrast to NKCC2, its expression is restricted to the medullary collecting duct and the glomerulus (15). Unlike NKCC2, the expression of NKCC1 is not limited to the kidney. NKCC1 is often referred to as the secretory cotransporter, because its expression is high in fluid-producing epithelia including the intestinal tract, salivary glands, submucosal glands, and the airway epithelia. However, pharmacological studies have identified furosemide-sensitive cotransporter activity in many cell types including hematopoietic cells, and airway and vascular smooth muscle. NKCC1 is believed to contribute to the regulation of cell volume in these nonepithelial cells (19). Of particular interest is the potential contribution of this transporter to the physiology of the endothelial cells, as they play a central role in many aspects of pneumonia and sepsis (20). Several studies suggest that NKCC1 regulates volume changes in these cells and that expression of NKCC1 by endothelial cells of the blood-brain barrier contributes to edema formation (21). It is therefore possible that similar expression of NKCC1 by lung endothelial cells can modulate the changes in microvascular permeability and thereby alter the course of inflammatory responses.
Mice lacking NKCC1 expression have been generated by several groups (22–26). The most readily observable phenotypes of the NKCC1–/– mice are their loss of hearing and inner ear function and male infertility. In addition, survival of the mice is limited on most genetic backgrounds, and generally those animals that do survive are considerably smaller in size. Most studies to date have been performed using mice of mixed genetic background. This has limited the use of these animals to study the role of NKCC1 during inflammatory responses, as the majority of these responses are sensitive to modifier genes (27, 28).
To circumvent this problem, in this study we generate congenic F1 NKCC1–/– mice and control littermates. We examine both the inflammatory response and the development of bacteremia and sepsis after lung infections with Klebsiella pneumoniae in these animals. We show that lack of the cotransporter reduces lung bacterial burden and dramatically reduces bacteremia and sepsis, which develops secondarily to this bacterial pneumonia. 
60% identity (10–12). NKCC2 (Slc12a1) encodes a cotransporter expressed almost exclusively in the kidney, where it is found in the cells of the ascending loop of Henle (13–15). Here it plays a critical role in the vectorial transport of ions (Na+, K+, and Cl–) from the glomerular ultra filtrate and hence is often referred to as the absorptive cotransporter. The fact that the improvement of the development of an intrapulmonary shunt after induction of acute lung injury was observed in dogs in which the kidneys had been surgically removed suggested that the beneficial effects of furosemide were not the result of blocking of this cotransporter (4). Similarly, in the premature infant, the improvement in gas exchange was more pronounced when furosemide was delivered as an aerosol, conditions under which the diuretic effects of this agent were limited (5). Thus, both of these studies suggested that the beneficial effects of furosemide in models and patients with acute lung injury were the result of inhibition of the second transporter NKCC1.
RESULTS
Top
ABSTRACT
RESULTS
DISCUSSION
MATERIALS AND METHODS
REFERENCES
Inflammation in NKCC1 mice after infection with K. pneumoniae
NKCC1–/– mice and littermate genetic controls were used to study the impact of loss of the cotransporter on the ability of the mice to mount a response to bacterial infection. In all cases, the mice were congenic F1 animals generated by the intercross of 129/SvEv+/– female animals with C57BL/6+/– males. Mice were infected with K. pneumoniae, and the development of disease was assessed 48 h later (Fig. 1).
Mice were killed and the cells present in the airways were collected by bronchoalveolar lavage (BAL). As expected, the bacterial infection resulted in a dramatic increase in the number of cells present in the BAL fluid (BALF). However, the number of cells present in the BALF collected from NKCC1–/– animals was significantly higher (P = 0.002) than that collected from the normal littermates. Cells present in the BAL were stained and identified based on morphological criteria. As expected, the majority of the cells recovered were neutrophils, and this population was significantly increased (P = 0.003) in the NKCC1–/– mice.
|
|
or IL-1ß. However, a small but significant decrease (P = 0.004) in the level of IL-10 was observed in the NKCC1–/– mice. Overall, the levels of cytokines measured in the lungs of the NKCC1–/– and wild-type mice were remarkably similar given the difference in numbers of inflammatory cells in the airways. To determine the impact of this difference in the number of neutrophils present in the airways on the progression of the infectious response in the mice, a second set of experiments was performed. Again the mice were infected with 4 x 104 bacteria. After 48 h mice were killed, blood was collected by heart stick, the lungs were collected and homogenized and CFUs in both samples were determined (Fig. 2). High levels of bacteria were detected in the blood of the wild-type mice. In contrast, loss of NKCC1 provided profound protection from the development of bacteremia with an almost 10-fold decrease in CFUs. This protection corresponded to decreased bacterial count in the homogenates prepared from the lungs of the NKCC1–/– animals compared with those prepared from wild-type mice. However, the difference in lung burden was not as dramatic as the difference observed upon comparison of CFUs present in the blood.
|
|
|
|
, keratinocyte chemoattractant, or macrophage inflammatory protein 2, although a small difference was observed in IL-6 levels (Fig. 7).
|
|
|
|
Attachment of neutrophils to activated endothelial cells is a prerequisite for recruitment during inflammatory responses. Activation of the endothelial cells results in expression of P and E selectins, which recognize L selectin expressed by neutrophils. Influx of neutrophils into the airways correlates with increased expression of vascular cell adhesion molecule (VCAM)-1. To determine whether the increased numbers of neutrophils could reflect altered induction of VCAM on endothelial cells, lungs were treated with LPS, and expression of VCAM was quantitated by Western blot analysis 6 h later (Fig. 9). As expected, LPS treatment results in an increase in levels of this protein. However, this change was observed in both NKCC1–/– and mutant mice and the magnitude of the change was similar in the two groups of animals.
Alterations in pulmonary vascular permeability in NKCC1–/– and wild-type mice after exposure to LPS
RNA expression analysis indicated that endothelial cells express NKCC1 (Fig. 4). Expression was easily detected both on a mouse endothelial cell line and on primary endothelial cell cultures, which we prepared from the mouse lung. Endothelial cells play a critical role both in sequestration of leukocytes and regulation of the permeability barrier of the pulmonary vasculature. Given the increased trafficking of cells into the airway and similarity in total numbers of neutrophils, we expected that the NKCC1–/– mice would display a similar or increased change in vascular permeability in the lung compared with their controls. To address this question, mice were treated with LPS to induce an inflammatory response (Fig. 10). Serum proteins were labeled by i.v. injection of animals with Evan's blue. At various times after exposure to LPS, mice were killed, the pulmonary vascular system was flushed with PBS, and the amount of dye present in the tissue was determined. In all cases the level of dye observed in control animals treated with vehicle was subtracted from the OD obtained from the LPS- treated animals. As expected, increased protein extravasation is observed 3 h after exposure to LPS. Surprisingly, the amount of serum protein present in the NKCC1–/– lungs was significantly decreased compared with that observed in wild-type animals (Fig. 10 A). Even at 6 h, when the edema is beginning to resolve, the difference between the two groups of animals persisted, suggesting that NKCC1 contributes to the changes in lung pulmonary vascular permeability during inflammation. To further address this point, we examined the lung wet to dry weight ratio of NKCC1–/– and control animals 3 h after instillation of LPS. This ratio was significantly reduced in the NKCC1–/– animals (Fig. 10 B).
|
DISCUSSION |
|---|
|
TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In our studies, we found that in the absence of NKCC1 expression colonization of the lungs with K. pneumoniae was reduced approximately threefold. Absence of the cotransporter had an even more dramatic effect on the development of bacteremia and sepsis. Although wild-type mice became moribund during the course of the experiment and were required to be killed within 48 h of intratracheal inoculation, the majority of the NKCC1–/– mice remained surprisingly healthy and active throughout the duration of the experiment. It is not clear at this point whether this dramatic difference in the development of hypothermic sepsis in the NKCC1–/– mice simply reflects the decreased bacterial burden in the lungs or whether other aspects of NKCC1 function also contribute to bacterial dissemination from the lung into the bloodstream.
It is unlikely that expression of NKCC1 on cells of hematopoietic origin contributes to the decreased morbidity of the NKCC1–/– mice. Levels of expression of NKCC1 detected by quantitative PCR are very low in neutrophils and macrophages. Consistent with this, no difference was observed in the ability of NKCC1-deficient neutrophils to kill K. pneumoniae in vitro or in the induction of a respiratory burst in response to PMA stimulation. Additionally, the study of bone marrow chimeras lacking NKCC1 on cells of hematopoietic lineages showed normal recruitment and localization of NKCC1–/– neutrophils when the lung expressed NKCC1. Increased numbers of neutrophils were collected by BAL from both K. pneumoniae–infected NKCC1–/– mice and NKCC1–/– mice treated with LPS. This indicates that the number of neutrophils that had both left the circulation and moved through the endothelial and epithelial tight junctions was increased in these animals. In contrast, little difference was seen in the number of neutrophils present in total lung homogenates from the LPS-challenged mice as determined by examination of myeloperoxidase levels. The most likely explanation for this is that change in expression of the cotransporter alters the pattern of neutrophil trafficking in the lung. It is possible that increased access to the airways results in enhanced clearance of K. pneumoniae and hence the decreased bacterial load.
NKCC1 is expressed by airway epithelial cells where it contributes to the formation of the airway surface liquid (30). NKCC1, located at the basolateral surface of the epithelium, works in tandem with cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl– channels to transport Cl– across many secretory epithelia. In this regard it is intriguing that cystic fibrosis patients, who also have a disturbance in Cl– secretion, have low incidence of sepsis despite high lung bacterial burdens. Staphylococcus aureus sepsis has not been described in CF patients, nor have invasive Pseudomonas infections been observed. One could speculate that the high levels of neutrophils in the CF lung also mimic what is observed in the NKCC1–/– mice. However, in the case of the CF patient, other factors and/or functions of CF render these recruited cells less effective at clearing the bacteria than appears to be the case in the NKCC1–/– mouse. For example, it has been suggested that CFTR directly regulates the activity of the epithelial sodium channel and thus that loss of CFTR results in Na+ hyperabsorption and dehydration of the airway surface of CF patients. These alterations would counteract the beneficial effects of increased neutrophil recruitment into alveolar spaces in a manner that does not compromise barrier function. This could provide an explanation of why these patients, similar to the NKCC1–/– mice, appear protected from sepsis, but unlike the NKCC1–/– mice, have increased lung bacterial burdens.
The relatively high expression of NKCC1 on endothelial cells and the critical role of these cells in regulation of fluid flux into lung tissues raises the possibility that this may represent the key cell type responsible for the increased survival of the NKCC1–/– mice with pneumonia. Fluid flux into the alveolar spaces is regulated by the lung capillary barrier and compromise of the integrity of this barrier leads to edema and intrapulmonary shunting. Altered endothelial function is the principal reason for failure of the barrier (31). It is interesting to speculate that increases in permeability of the barrier require contraction of the endothelial cell and a change in cell shape, and that these events are accompanied by a change in cell volume. It is possible that NKCC1 is required for these changes and when cells lack the cotransporter these changes are compromised. If this is the case, our failure to observe a decrease in edema formation in K. pneumoniae–induced peritonitis would suggest that this function of NKCC1 is critical only in some capillary beds. An alternative model is suggested by studies which have demonstrated a role for NKCC1 in vascular tone (32). It is possible that decreased vascular pressure decreases edema formation in the lung. Again, in this model the contribution of NKCC1 to venous tone would be limited to specific vascular beds. The development of mouse lines in which loss of expression of NKCC1 is limited to specific tissues should help to distinguish between these various models.
In summary, we show here that loss of NKCC1 expression dramatically alters the course of bacterial pneumonia. Importantly, mice lacking the NKCC1 cotransporter were protected from sepsis and bacteremia. The impact of loss of NKCC1 was observed primarily in the lung. In contrast, the NKCC1–/– mice tended toward more severe disease when K. pneumoniae was introduced into the peritoneal cavity. Collectively, our results suggest that inhibitors specific for NKCC1 might provide a novel means of limiting sepsis in individuals with bacterial pneumonia. Given the profound impact of loss of NKCC1 on disease progression, it will be of great interest to more precisely define the mechanism underlying the attenuation of disease upon loss of NKCC1 activity. This should be possible with the development of culture conditions that allow in vitro study of endothelial barrier function of cells prepared from NKCC1–/– and control animals, and the generation of mouse lines in which the loss of expression of NKCC1 can be regulated in a cell type–specific manner.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Bacteria.
Mouse virulent strain K. pneumoniae 43816, serotype 2, from the American Type Culture Collection were aerobically grown in nutrient broth for 2 h at 37°C. Bacteria numbers were estimated by measuring the absorbance at 600 nm (1 OD = 3 x 108 bacteria/ml) and exact number used in each experiment determining CFUs after plating an aliquot onto a nutrient agar plate.
K. pneumoniae–induced lung inflammation.
Mice were anesthetized, placed in an upright position, and suspended by the incisors over a supporting wire. The tongue was extended, and 4 x 104 CFU of K. pneumoniae in 50 µl PBS was delivered onto the vocal cords.
Assessment of lung inflammatory cells.
48 h postinfection with K. pneumoniae and 6 h after LPS challenge, the lungs were lavaged five times with Hanks' balance salt solution. The recovered BALF was pooled, and a 50-µl aliquot was reserved for determination of total cell counts with a hemacytometer. For differential cell counts, cytospin slides were prepared, stained using a Hema-3 staining kit (Fisher Scientific), and >200 leukocytes were identified based on morphological criteria per slide from each sample.
Assessment of K. pneumoniae in blood and lung tissues.
48 h postinfection with K. pneumoniae, mice were anesthetized and blood samples were obtained by cardiac puncture. Lungs were removed after pulmonary perfusion with PBS/5 mM EDTA and homogenized in a glass grinder with 2 ml of homogenate buffer (PBS with 1% Triton X-100) containing protease inhibitor tablet (Roche Diagnostics). Aliquots of the suspensions were plated onto LP plates, and CFUs were determined.
Cytokine and chemokine analysis postinflammation.
After centrifugation of lung homogenates, supernatants were passed through a 0.45-µm syringe-driven filter unit to remove bacteria. TNF-, IL-1ß, IL-10, KC, and MIP-2 levels in supernatants and in BALF were determined by commercial ELISA kits (R&D Systems) according to the manufacturer's instructions.
Preparation of specific immune serum and opsonization of bacteria.
Specific immune serum was prepared as described previously (33). Bacteria (107) were suspended in 1 ml of HBSS containing 1 mM CaCl2, 0.5 mM MgCl2 and 1 mM Hepes with 5% specific immune serum for 15 min at 37°C with end-to-end rotation before use.
Preparation of neutrophils and neutrophil-killing assays.
Bone marrow was collected from mouse femurs and tibias, and neutrophils were isolated by density gradient centrifugation through 2 Histopaque layers (density 1.083 g/ml and 1.119 g/L; Sigma-Alrich). Preparations were > 90% pure as determined by staining with the Hema-3 staining kit (Fisher Scientific) and examination of cell morphology. Assessment of bacterial killing by neutrophils was performed as previously described (34, 35).
Measurement of superoxide production.
Superoxide production was monitored by measurement of the superoxide dismutase–inhibitable cytochrome C reduction in a modification of methods described by Pick and Mizel (36). Superoxide produced by neutrophils was calculated from the extinction coefficient of cytochrome C by the formula 
E550 = 2103 M–1cm–1. The results were expressed as nanomoles of reduced cytochrome C.
LPS-induced lung inflammation.
Anesthetized mice received 0.3 mg/kg of LPS (from Escherichia coli, Sigma-Aldrich) in 50 µl PBS intratracheally. 6 h later, mice were killed and harvested for assessment of airway inflammatory cells, mediator levels, and MPO activity.
MPO assay.
MPO activity in lung tissues was measured 6 h after LPS exposure as described previously (37–41). MPO activity was quantified by extrapolation from the standard curve.
Lung permeability assay.
Lung capillary permeability was determined by Evan's blue dye extravasation (42). 5 h after intratracheal challenge with LPS (1 mg/kg), mice received 10 mg/ml solution of Evan's blue dye i.v. (100 mg/kg). 1 h later, mice were killed and lungs were harvested after perfusion with HBSS and incubated in 2 ml of formamide at 55°C for 48 h. The absorbance of the Evan's blue extracts was spectrophotometrically measured at 620 and 740 nm. The following formula was used to correct for the presence of heme pigments: A620 (corrected) = (1.426 x A740 + 0.030).
Lung wet-to-dry weight ratio measurement.
Mice were treated with LPS and 3 h later killed and whole lungs were excised and blotted dry and weighed. Lung dry weights were recorded after lung samples were dried in the oven for 3 d at 65°C. The lung wet-to-dry weight ratio was calculated by dividing the wet weight by the dry weight.
Isolation of pulmonary endothelial cells.
Endothelial cells were isolated by magnetic cell sorting of labeled endothelial cells using a MACS separation unit (Miltenyi Biotec) as described previously by Marelli-Berg et al. (43). Endothelial cells were plated out onto 1% gelatin- (type B from bovine) coated tissue culture plates and cultured until confluent. At confluence, endothelial cells were characterized and RNA was prepared.
Quantitative RT-PCR.
Total RNA was isolated using RNazol B (Teltest). Reverse transcription of RNA to cDNA for quantitative RT-PCR was performed using a high-capacity cDNA archive kit (Applied Biosystems) according to the manufacturer's instructions. Primers and probes were purchased from Applied Biosystems. All reactions were performed with TaqMan PCR Universal Master Mix (Applied Biosystems) using the Applied Biosystems 7900 HT Fast Real-Time PCR System. Reactions were performed in quadruplicate in a 20 µl final volume reaction. Expression levels of genes of interest were normalized to 18S RNA. Data were analyzed using the comparative CT method as described by Applied Biosystems.
Lung histology.
Mice received 0.3 mg/kg of LPS or vehicle intratraceally as described above. Mice were killed 12 h later and lungs were inflated, removed, and fixed in 2% paraformaldehyde/4% glutaraldehyde in Karlsson and Schultz sodium phosphate buffer at 4°C overnight. The left lobes were processed and embedded in paraffin wax. 4-µm-thick sections were cut and stained with hematoxylin and eosin. The other lobes were diced into small pieces and fixed again in glutaraldehyde fixative for 24 h. Subsequently, lung samples were postfixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and Epon infiltrated in an automated Reichert Lynx EM tissue processor. Specimens were embedded in Epon 812 and polymerized overnight at 65°C. 0.5-µm-thick sections were cut and stained with 1% Toluidine blue-O in 1% sodium borate. Slides were scanned with an Aperio ScanScope scanner (Aperio Technologies, Inc.) at 20x power to produce a high resolution digital image. Digital images were then analyzed using ImageScope viewing software (Aperio Technologies, Inc.), and neutrophils were counted in a blinded fashion.
K. pneumoniae–induced peritoneal inflammation.
Mice received 0.5% Evan's blue dye (Sigma-Aldrich) i.v. (10 mg/kg) followed by i.p injection of 106 CFU K. pneumoniae. 1 h after infection, mice were killed and the peritoneal cavities were lavaged with 4 ml of cold PBS. The number of peritoneal exudate cells in the peritoneal lavage fluid was determined by cell counts using a hemacytometer, and cellular differential were performed on cytospun slides. CFUs were determined after 18 h of incubation at room temperature on agar plates. Plasma protein extravasation into the peritoneal cavity was determined by measuring the absorbance of Evan's blue dye in the cell-free supernatants.
Western blotting.
Lung lysates were prepared in lysis buffer. Samples (100 µg) were electrophoresed on 10% SDS-PAGE gels, and the proteins were electrophoretically transferred to PVDF membrane. Membranes were blocked in 5% nonfat milk in TBS with 0.1% Tween 20 for 1 h at room temperature and incubated overnight at 4°C with polyclonal anti–mouse VCAM-1 (R & D Systems, Inc.) at a final concentration of 0.1 µg/ml. Horseradish peroxidase–conjugated goat anti–mouse immunoglobulin G (Santa Cruz Biotechnology) was used as an appropriate secondary antibody. The blot was stripped and reprobed with anti-GAPDH (Imgenex) at a 1:5,000 dilution.
Generation of chimeric mice.
Chimeric mice were generated using a previously described bone marrow transplantation method with modifications (44). In brief, whole bone marrow cells were harvested from femurs and tibias of 8-wk-old donor mice. Bone marrow cells were washed once, filtered through Miracloth (Calbiochem), and resuspended in PBS. Recipient mice were lethally irradiated with two exposures to 5 Gy 3 h apart. Immediately after the second exposure of irradiation, donor bone marrow cells were injected i.v into lethally irradiated recipient mice. Chimeric animals were used 6–8 wk after bone marrow transplantation.
Statistical analysis.
Data are presented as mean ± SEM. Statistical comparisons between groups of mice were made by two-tailed Student's t-test. P < 0.05 was considered statistically significant.
|
Acknowledgments |
|---|
The authors would like to thank A. Latour for assistance with animal genotyping, B. Bagnell and V. Madden for assistance with microscopy, and L. Jania for statistical analysis.
The authors have no conflicting financial interests.
Submitted: 7 June 2006
Accepted: 24 April 2007
|
REFERENCES |
|---|
|
TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Wheeler, A.P., and G.R. Bernard. 1999. Treating patients with severe sepsis. N. Engl. J. Med. 340:207–214.
2 Hudson, L.D., and K.P. Steinberg. 1999. Epidemiology of acute lung injury and ARDS. Chest. 116:74S–82S.[CrossRef]
3 Hasleton, P.S., and T.E. Roberts. 1999. Adult respiratory distress syndrome—an update. Histopathology. 34:285–294.[CrossRef][Medline]
4 Ali, J., W. Chernicki, and L.D. Wood. 1979. Effect of furosemide in canine low-pressure pulmonary edema. J. Clin. Invest. 64:1494–1504.[Medline]
5 Prabhu, V.G., M. Keszler, and R. Dhanireddy. 1997. Pulmonary function changes after nebulised and intravenous frusemide in ventilated premature infants. Arch. Dis. Child. Fetal Neonatal Ed. 77:F32–F35.
6 Haas, M. 1994. The Na-K-Cl cotransporters. Am. J. Physiol. 267:C869–C885.[Medline]
7 Russell, J.M. 2000. Sodium-potassium-chloride cotransport. Physiol. Rev. 80:211–276.
8 O'Grady, S.M., H.C. Palfrey, and M. Field. 1987. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues. Am. J. Physiol. 253:C177–C192.[Medline]
9 Haas, M., and B. Forbush III. 2000. The Na-K-Cl cotransporter of secretory epithelia. Annu. Rev. Physiol. 62:515–534.[CrossRef][Medline]
10 Delpire, E., M.I. Rauchman, D.R. Beier, S.C. Hebert, and S.R. Gullans. 1994. Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells. J. Biol. Chem. 269:25677–25683.
11 Payne, J.A., J.C. Xu, M. Haas, C.Y. Lytle, D. Ward, and B. Forbush III. 1995. Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. J. Biol. Chem. 270:17977–17985.
12 Park, J.H., and M.H. Saier. 1996. Phylogenetic, structural and functional characteristics of the Na-K-Cl cotransporter family. J. Membr. Biol. 149:161–168.[CrossRef][Medline]
13 Gamba, G., A. Miyanoshita, M. Lombardi, J. Lytton, W.-S. Lee, M.A. Hediger, and S.C. Hebert. 1994. Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J. Biol. Chem. 269:17713–17722.
14 Igarashi, P., G.B.V. Heuvel, J.A. Payne, and B. Forbush III. 1995. Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter. Am. J. Physiol. 269:f405–f418.[Medline]
15 Payne, J.A., and B. Forbush III. 1994. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney. Proc. Natl. Acad. Sci. USA. 91:4544–4548.
16 Lytle, C, J.-C. Xu, D. Biemesderfer, and B. Forbush III. 1995. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am. J. Physiol. 269:c1496–c1505.[Medline]
17 Delpire, E., M.I. Rauchman, D.R. Beier, S.C. Hebert, and S.R. Gullans. 1994. Molecular cloning and chromosome localization of a putative basolateral Na+-K+-2Cl– cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells. J. Biol. Chem. 269:25677–25683.
18 Xu, J.-C., C.Y. Lytle, T.T. Zhu, J.A. Payne, E. Benz Jr., and B. Forbush III. 1994. Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. Proc. Natl. Acad. Sci. USA. 91:2201–2205.
19 O'Donnell, M.E. 1993. Role of Na-K-Cl cotransport in vascular endothelial cell volume regulation. Am. J. Physiol. 264:C1316–C1326.[Medline]
20 Aird, W.C. 2003. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 101:3765–3777.
21 O'Donnell, M.E., L. Tran, T.I. Lam, X.B. Liu, and S.E. Anderson. 2004. Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J. Cereb. Blood Flow Metab. 24:1046–1056.[CrossRef][Medline]
22 Dixon, M.J., J. Gazzard, S.S. Chaudhry, N. Sampson, B.A. Schulte, and K.P. Steel. 1999. Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice. Hum. Mol. Genet. 8:1579–1584.
23 Delpire, E., J. Lu, R. England, C. Dull, and T. Thorne. 1999. Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter. Nat. Genet. 22:192–195.[CrossRef][Medline]
24 Flagella, M., L.L. Clarke, M.L. Miller, L.C. Erway, R.A. Giannella, A. Andringa, L.R. Gawenis, J. Kramer, J.J. Duffy, T. Doetschman, et al. 1999. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J. Biol. Chem. 274:26946–26955.
25 Pace, A.J., E. Lee, K. Athirakul, T.M. Coffman, D.A. O'Brien, and B.H. Koller. 2000. Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl- cotransporter. J. Clin. Invest. 105:441–450.[Medline]
26 Pace, A.J., V.J. Madden, O.W. Henson Jr., B.H. Koller, and M.M. Henson. 2001. Ultrastructure of the inner ear of NKCC1-deficient mice. Hear. Res. 156:17–30.[CrossRef][Medline]
27 Thach, D.C., T. Kimura, and D.E. Griffin. 2000. Differences between C57BL/6 and BALB/cBy mice in mortality and virus replication after intranasal infection with neuroadapted Sindbis virus. J. Virol. 74:6156–6161.
28 Weinberg, J.B., M.L. Lutzke, R. Alfinito, and R. Rochford. 2004. Mouse strain differences in the chemokine response to acute lung infection with a murine gammaherpesvirus. Viral Immunol. 17:69–77.[CrossRef][Medline]
29 Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23:197–223.[CrossRef][Medline]
30 Grubb, B.R., A.J. Pace, E. Lee, B.H. Koller, and R.C. Boucher. 2001. Alterations in airway ion transport in NKCC1-deficient mice. Am. J. Physiol. Cell Physiol. 281:C615–C623.
31 Dudek, S.M., and J.G. Garcia. 2001. Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91:1487–1500.
32 Meyer, J.W., M. Flagella, R.L. Sutliff, J.N. Lorenz, M.L. Nieman, C.S. Weber, R.J. Paul, and G.E. Shull. 2002. Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na(+)-K(+)-2Cl(-) cotransporter. Am. J. Physiol. Heart Circ. Physiol. 283:H1846–H1855.
33 Mancuso, P., T.J. Standiford, T. Marshall, and M. Peters-Golden. 1998. 5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae. Infect. Immun. 66:5140–5146.
34 Rubin, B.B., G.P. Downey, A. Koh, N. Degousee, F. Ghomashchi, L. Nallan, E. Stefanski, D.W. Harkin, C. Sun, B.P. Smart, et al. 2005. Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity. J. Biol. Chem. 280:7519–7529.
35 Dziarski, R., K.A. Platt, E. Gelius, H. Steiner, and D. Gupta. 2003. Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)-deficient mice. Blood. 102:689–697.
36 Pick, E., and D. Mizel. 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods. 46:211–226.[CrossRef][Medline]
37 Mehrad, B., R.M. Strieter, T.A. Moore, W.C. Tsai, S.A. Lira, and T.J. Standiford. 1999. CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis. J. Immunol. 163:6086–6094.
38 Manderscheid, P.A., R.P. Bodkin, B.A. Davidson, E. Jensen, T.A. Russo, and P.R. Knight. 2004. Bacterial clearance and cytokine profiles in a murine model of postsurgical nosocomial pneumonia. Clin. Diagn. Lab. Immunol. 11:742–751.[CrossRef][Medline]
39 Shao, C., J. Qu, L. He, Y. Zhang, J. Wang, Y. Wang, H. Zhou, and X. Liu. 2005. Transient overexpression of gamma interferon promotes Aspergillus clearance in invasive pulmonary aspergillosis. Clin. Exp. Immunol. 142:233–241.[CrossRef][Medline]
40 Power, M.R., Y. Peng, E. Maydanski, J.S. Marshall, and T.J. Lin. 2004. The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice. J. Biol. Chem. 279:49315–49322.
41 Jain-Vora, S., A.M. LeVine, Z. Chroneos, G.F. Ross, W.M. Hull, and J.A. Whitsett. 1998. Interleukin-4 enhances pulmonary clearance of Pseudomonas aeruginosa. Infect. Immun. 66:4229–4236.
42 Wang, E., N. Ouellet, M. Simard, I. Fillion, Y. Bergeron, D. Beauchamp, and M.G. Bergeron. 2001. Pulmonary and systemic host response to Streptococcus pneumoniae and Klebsiella pneumoniae bacteremia in normal and immunosuppressed mice. Infect. Immun. 69:5294–5304.
43 Marelli-Berg, F.M., E. Peek, E.A. Lidington, H.J. Stauss, and R.I. Lechler. 2000. Isolation of endothelial cells from murine tissue. J. Immunol. Methods. 244:205–215.[CrossRef][Medline]
44 Fabre, J.E., J.L. Goulet, E. Riche, M. Nguyen, K. Coggins, S. Offenbacher, and B.H. Koller. 2002. Transcellular biosynthesis contributes to the production of leukotrienes during inflammatory responses in vivo. J. Clin. Invest. 109:1373–1380.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
, 
, P < 0.01 relative to alveolar macrophage mRNA. Statistical analysis was performed using one-way ANOVA with Tukey's Multiple Comparisons posttest.
NKCC–/–), whereas NKCC1+/+ mice received marrow from NKCC1–/– mice (NKCC–/–
