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* From the UBC, Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, BC, Canada.
Correspondence to: Stephan F. van Eeden, MD, Pulmonary Research Laboratory, St Paul's Hospital, 1081 Burrard St, Vancouver, BC V6Z 1Y6, Canada; e-mail: svaneeden{at}prl.pulmonary.ubc.ca
Polymorphonuclear leukocytes (PMNs) are essential for host defense, but they also mediate tissue injury and have been implicated in the pathogenesis of several important diseases, including those that may trigger acute lung injury and ARDS.1 2 In these conditions, activated PMNs accumulate in lung microvessels and cause endothelial damage, increased vascular permeability, and produce tissue injury.3 The PMNs that mediate these changes come from either those already in the vascular space where they either circulate or marginate along vessel walls, or those released from the bone marrow into the vascular space.4
The release of PMNs from the bone marrow is an important component of the systemic response to an inflammatory stimulus. Normally only fully differentiated PMNs enter the circulation, but stimulation of the bone marrow during an inflammatory reaction results in the release of more immature PMNs into the circulation.4 Immature PMNs harvested from the bone marrow have been shown to be less deformable, have reduced motility, and are less phagocytic if compared with mature cells in peripheral blood.5 Studies from our laboratory have shown that PMNs newly released from the bone marrow express higher levels of L-selectin than their counterparts in the circulation, a molecule that contributes to the recruitment of PMNs to a site of inflammation.6 The role of PMNs newly released from the marrow in the pathogenesis of the acute lung injury is unclear. This study explores the possible role of this population of younger PMNs in the pathogenesis of acute lung injury associated with sepsis.
Sequestration and Migration of Newly Released PMNS in Lung Microvessels
The thymidine analogue 5'bromo-2-deoxyuridine (BrdU) was used to label dividing myeloid cells in the bone marrow, and the release of these labeled cells (PMNBrdU) into the circulation was used as a marker of PMNs newly released from the bone marrow.7 8 New Zealand white rabbits were given BrdU (100 mg/kg IV over 15 min) 24 h prior to stimulating the bone marrow to release the PMNBrdU. To determine the sequestration and migration of PMNBrdU in the lung, a focal pneumococcal pneumonia (Streptococcus pneumonaie 5 x 108 organisms), a focal pneumonia followed by bacteremia (5 x 108 organisms) 4 h later, or granulocyte colony-stimulating factor (G-CSF) (12.5 µg/kg IV; Amgen Inc; Thousand Oaks, CA) was given 24 h following the labeling. Animals were observed for 8 h, killed, and the lungs were processed for morphometric analysis as previously described.7 BrdU-labeled PMNs in lung tissue were identified using immunohistochemistry and enumerated using standard morphometric techniques.7 Figure 1 (top, a) shows the percentage of PMNBrdU sequestered in pulmonary capillaries compared with circulating blood. Significantly more PMNBrdU sequestered in pulmonary capillaries in the untreated contralateral lung and in the pneumonic lung region compared with circulating blood both in the focal pneumonia (p < 0.01), bacteremic pneumonia, and G-CSF-treated groups (p < 0.05). This showed that PMNs newly released from the bone marrow preferentially sequester in pulmonary capillaries, suggesting that these younger PMNs have a prolonged transit time through the lung. PMNs need to deform to cross the pulmonary capillary bed because of the discrepancy between the size of PMNs and pulmonary capillary segments.9 Preferential sequestration of younger PMNs in lung microvessels suggests that these PMNs are less deformable than their mature circulating counterparts. Differences in their response to cell activation with regards to adhesiveness,6 10 size, and deformability could also contribute to their preferential sequestration in the lung. The pattern of sequestration of PMNBrdU in pulmonary capillaries following G-CSF treatment was similar to untreated regions in the pneumonia groups, suggesting that the phenotypic and functional characteristics of PMN release from the bone marrow by these stimuli are similar. It is also consistent with G-CSF being an important mediator of the bone marrow response in pneumonia.
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In Vitro Deformability and Chemotaxis of Newly Released PMNs
The deformability and chemotactic ability of newly released PMNs were determined 8 h following stimulation of the bone marrow with saline solution (control), lipopolysaccharide (LPS) (10 µg/kg IV), G-CSF (12.5 µg/kg subcutaneous), a focal pneumococcal pneumonia (S pneumonaie 5 x 108 organisms), and a 4-h focal pneumonia followed by bacteremia (5 x 108 organisms). For deformability studies, leukocyte-rich plasma prepared from whole blood was filtered in vitro according to a modified filtration method.12 Briefly, leukocytes in a 20-mL polypropylene syringe were filtered through a 5-µm pool-size polycarbonate filter (Poretics; Livermore, CA) using a syringe infusion pump (Pump 22; Harvard Apparatus; Millis, MA) that provides a constant-flow rate of 1 mL/min solution across the filter. All filter syringes and tubing were coated with 5% human serum albumin (USP Plasmin-5 Miles) for 2 h before filtration. Hydrostatic pressure was continuously monitored upstream from the filter using a pressure transducer (Validyne Engineering; Northridge, CA) connected to a recording system. The pressure sensing system was calibrated using a water manometer under conditions of no flow before each filtration. Cell viability after filtration was 98% as tested by trypan blue exclusion. Samples for analysis were collected at the plateau pressure (steady-state conditions). For chemotaxis studies, PMNs were purified and plated on 5-µm pool-size polycarbonate filters in a Boyden chamber using rabbit recombinant interleukin-8 (2 µg/mL; Genetech Inc; San Francisco, CA) as chemoattractant.
The percentage of PMNBrdU in samples was determined using immunocytochemistry as previously described.7 8 Results are expressed as the percentage retention of PMNBrdU in filters. Figure 1 (bottom, b) shows the retention of PMNBrdU in filters. Both in the filtration and chemotaxis experiments, significantly more PMNBrdU was retained in filters in the pneumonia, LPS, and G-CSF groups compared with the control group (p < 0.01) consistent with younger PMNs released from the bone marrow by an inflammatory stimulus being less deformable and less chemotactic than their circulating counterparts. Together, these in vitro studies support the in vivo observations that younger PMNs show preferential sequestration in lung microvessels and are slow to migrate into the inflammatory site.
Conclusion
In studies of different inflammatory conditions, peripheral blood PMNs have been shown to be a heterogeneous population of cells. In this study, we showed heterogeneity of circulating PMN maturity. Younger PMNs newly released from the bone marrow by inflammatory stimuli are less deformable with a reduced chemotactic ability. These abnormalities are similar to findings of PMNs harvested from the bone marrow5 11 and result in preferential sequestration of these PMNs in lung microvessels and slow migration into the inflammatory site. These maturity-related abnormalities of younger PMNs could compromise their ability to eradicate infection but may also cause damage to adjacent endothelium if these PMNs trapped in lung microvessels are activated by circulating inflammatory mediators.10 The contribution of this subpopulation of PMNs in the pathogenesis of acute lung injury needs to be determined.
Footnotes
Supported by the Medical Research Council of Canada (grant 4219).
References
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  C.-H. Shih, B. A. Whalen, Y. Goto, J. C. Hogg, and S. F. van Eeden Flow cytometric method for enumeration and characterization of newly released polymorphonuclear leukocytes from the bone marrow using 5'-bromo-2'-deoxyuridine Am J Physiol Cell Physiol, September 1, 2005; 289(3): C757 - C765. [Abstract] [Full Text] [PDF]
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