Leukocyte adhesion deficiency syndrome

The biology of nitric oxide (NO) has become a very interesting story in inflammatory pulmonary disease, with many complexities. Its role in the initiation and evolution of acute lung injury and in neutrophil trafficking has proven particularly curious, and, at each turn, there have been interesting surprises. The study by Razavi and colleagues in this issue of the Journal (pp. 227-233) elegantly addresses the effect of inducible nitric oxide synthase (iNOS)-generated NO on several steps in the process of neutrophil trafficking in the lungs during sepsis, including neutrophil sequestration evaluated by in vivo video microscopy and neutrophil migration into the airspaces using bronchoalveolar lavage (1). The investigators make the interesting observation that during sepsis induced by cecal ligation and puncture, fewer neutrophils sequester in the pulmonary capillaries of mice deficient in iNOS-generated NO than in wild-type mice, but more neutrophils migrate, suggesting that NO is required for maximal sequestration of neutrophils within the lung capillaries but acts to slow or attenuate neutrophil migration into the airspaces.

These data raise several interesting points. First, they clearly underline the importance of examining the roles of important signaling molecules in each step of the process through which neutrophils emigrate. The mechanisms through which neutrophils recognize an inflammatory site in the lungs, slow down and stop, adhere to the endothelium, migrate along the endothelial surface, and transmigrate between or through endothelial cells and through the alveolar walls require individual attention (2). Even in the signaling that modulates cytoskeletal events, NO appears to play varying roles in the stiffening that likely mediates sequestration of neutrophils compared with the crawling that occurs during migration. Approaches such as the ones taken by these authors begin to sort out these processes in the whole animal and emphasize the critical role bioimaging can play. For example, measuring the capillary transit times of neutrophils may provide valuable information about whether NO mediated the recognition and stopping of neutrophils or the transition to adhesion and transmigration.

Second, these data may suggest a potential role for NO in the maturation and release of neutrophils from the bone marrow. Sepsis induced by cecal ligation and puncture induces bone marrow release, as demonstrated by the increase in circulating and sequestered neutrophils (1). Despite fewer numbers of neutrophils sequestering in the iNOS^sup -/-^ mice, the circulating neutrophil counts are similar in both genotypes and actually tend to be lower in the iNOS^sup -/-^ mice. The lack of an increase in the circulating counts in the iNOS^sup -/-^ mice with decreased sequestration may suggest a defect in bone marrow release in the absence of iNOS. In light of NO's recognized contribution to cell-cell and cell-matrix adhesion and to cytoskeletal dynamics, which are certain to be important in this process as well as in neutrophil recruitment (3-5), NO may modulate neutrophil trafficking from the bone marrow.

Third, these data raise exciting questions about the role of NO in neutrophil migration. One explanation for the increased migration in the absent of iNOS-generated NO might be proliferation of bacteria in the absence of NO and hence a greater stimulus (6,7). The authors' observation, however, that iNOS^sup -/-^ neutrophils demonstrate enhanced migration across endothelial cell monolayers in response to exogenous cytokines makes this possibility less likely. These data invite studies about the role of NO in neutrophils during intracellular signaling, adhesion molecule expression and function, chemokine signaling, oxidant production, cytoskeletal rearrangements, and crawling that occur during migration.

Whether the iNOS expressed by neutrophils or lung parenchymal cells is the source of the NO regulating sequestration and migration was determined using lethal irradiation and reconstitution to generate mice deficient in iNOS in either the recipient or the stem cell-derived hematopoielic cells. This technique is becoming a common approach to addressing this question for many molecules. Concerns are injury to the lungs and other organs induced by the procedure and the genotype of the alveolar macrophages, both of which depend on the dose of radiation, the length of the reconstitution, and whether the genotype of the mutant cells alters their responses. Razavi and colleagues (1) used this approach elegantly to demonstrate that iNOS in the stem cell-derived population, most likely the neutrophils, is responsible for the defects in both sequestration and migration, whereas iNOS-derived NO generated by recipient lung endothelial or other parenchymal cells plays no observable role. Their data examining neutrophil migration across cultured pulmonary microvascular endothelial cells further support these observations that NO derived from neutrophils and not from endothelial cells acts to delay or depress neutrophil transendothelial migration.

While the authors have chosen appropriately to temper their interpretation to suggest that the iNOS in neutrophils and not lung parenchymal cells is modulating these effects on neutrophil trafficking during sepsis, an editorialist's license to fantasize leads me to point out that the effects of iNOS deficiency in only the stem cells appear far greater than the effects of iNOS deficiency in the whole animal (1). For example, the number of sequestered neutrophils in the iNOS^sup -/-^ mice was 20-40% less than the number in wild-type mice (see their Figure 3), whereas the number of iNOS^sup -/-^ neutrophils in wild-type mice was 70-80% less than the number of wild-type neutrophils (see their Figure 6B). Similarly, two- to threefold more neutrophils migrated into the airspace of the iNOS^sup -/-^ than wild-type mice (see Figure 5), compared with an increase of 8- to 10-fold in iNOS^sup -/-^ neutrophils reconstituted in wild-type mice (see Figure 6C). Furthermore, the increase in circulating neutrophils induced by sepsis was not significantly different between iNOS^sup -/-^ and wild-type mice, but was completely prevented when only the neutrophils were iNOS^sup -/-^ (compare Figure 1 with Figure 6A). Studies of this injury in iNOS^sup -/-^ mice reconstituted with iNOS^sup -/-^ stem cells and comparisons with uninjured reconstituted mice will help to confirm these observations that the completely iNOS^sup -/-^ mice have a milder phenotype than wild-type mice given iNOS^sup -/-^ stem cells. The data are very tantalizing, however, and appear to suggest that iNOS-generated NO in parenchymal cells of the recipient may have contrasting effects that balance the effects of NO produced by the neutrophils, when the integrated response of the intact animal is considered.

Razavi and colleagues (1) have clearly pointed out important concerns regarding how closely this model of sepsis and very minimal lung injury mimics sepsis and acute respiratory distress syndrome in patients, and regarding the controversies about the role of iNOS in murine compared with human neutrophils. These are valid and underline the need for translational studies, but should not decrease the importance of their observations and the need to understand their basis. These studies raise many important new questions, including the role of NO in facilitating neutrophil sequestration, attenuating migration, regulating bone marrow release and lifespan, and contributing to regulation of pulmonary vascular permeability (8). Their article makes a critical contribution in identifying varying roles of iNOS-generated NO in neutrophil trafficking during sepsis.

References

1. Razavi HM, Wang LF, Weicker S, Rohan M, Law C, McCormack DG, Mehta S. Pulmonary neutrophil infiltration in murine sepsis: Role of inducible nitric oxide synthase. Am J Respir Crit Care Med 2004;170: 227-233.

2. Doerschuk CM. Mechanisms of leukocyte sequestration in inflamed lungs. Microcirculation 2001;8:71-88.

3. Sato Y, Van Eeden SF, English D, Hogg JC. Pulmonary sequestration of polymorphonuclear leukocytes released from bone marrow in bacteremic infection. Am J Physiol 1998;275:L255-L261.

4. Sato Y, Walley KR, Klut ME, English D, D'yachkova Y, Hogg JC, van Eeden SF. Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression. Am J Respir Crit Care Med 1999;159:1469-1476.

5. Saito H, Lai J, Rogers R, Doerschuk CM. The mechanical properties of rat bone marrow and circulating neutrophils and their responses to inflammatory mediators. Blood 2002;99:2207-2213.

6. Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA 2000;97:8841-8848.

7. Pacelli R, Wink DA, Cook JA, Krishna MC, DeGraff W, Friedman N, Tsokos M, Samuni A, Mitchell JB. Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli. J Exp Med 1995;182:1469-1479.

8. Wang LE, Patel M, Razavi HM, Weicker S, Joseph MG, McCormack DG, Mehta S. Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 2002;165:1634-1639.

DOI: 10.1164/rccm.2405005

Conflict of Interest Statement: C.M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

CLAIRE M. DOERSCHUK, M.D.

Department of Pediatrics

Rainbow Babies and Children's Hospital and Case Western Reserve University

Cleveland, Ohio

Copyright American Thoracic Society Aug 1, 2004
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