Journal of Applied Physiology
Vol. 82, No. 5, pp. 1395-1396, May 1997

Invited Editorial on "Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes"

Gregory P. Downey

Division of Respiratory Diseases, Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

ARTICLE

REFERENCES

ARTICLE

NEUTROPHILIC POLYMORPHONUCLEAR LEUKOCYTES (neutrophils or PMN) serve a crucial physiological function in host defense as phagocytes. Paradoxically, in pathological conditions characterized by inflammatory injury such as myocardial infarction, stroke, and acute lung injury, control mechanisms fail, and neutrophils contribute to injury to host tissues by several mechanisms. Occlusion of capillaries by leukocytes leads to heterogeneity of perfusion, with total occlusion of some microvessels and increased perfusion to others. This has obvious deleterious effects, including defective delivery of O₂ and removal of CO₂ and other metabolic by-products. Additionally, PMN sequestered in microvascular beds adhere to the endothelium and emigrate from the vascular space into the interstitial tissues where they can be induced to release a number of toxic compounds, including reactive O₂ intermediates (ROI), proteolytic enzymes, lipid mediators, and cytokines that induce or promote injury to vicinal cells and potentiate the inflammatory response. Despite intense investigation over the last several decades, our knowledge of the events occurring within the microvasculature is incomplete.

During their brief sojourn through the body, PMN are subject to a variety of mechanical stresses in diverse environments. After differentiating in the bone marrow, mature PMN must squeeze through migration channels in the walls of the bone marrow sinuses to enter the bloodstream (12). Subsequently, PMN must repetitively negotiate disparate microvascular beds, including those in muscle, kidney, brain, heart, gastrointestinal tract, and lung. The size discrepancy between PMN (~7-8 µm in diameter) and capillaries (7.4 µm mean diameter with a range of 2-15 µm in the lung) mandates that PMN must repetitively deform during passage through these microvessels (5). The unique arrangement of capillaries in the lung exaggerates this situation, as it has been estimated that PMN must negotiate 50-100 capillary segments between the arterial and venous side of the pulmonary vascular bed (9). Reports from many laboratories (4, 2, 8, 16) have documented that the biophysical properties (stiffness or deformability) of leukocytes are a major determinant of their initial retention within microvascular beds: the stiffer (less deformable) the cell, the longer the microvascular transit time. Cellular deformability is determined in large part by the amount and spatial distribution of the actin cytoskeleton (16). The subsequent phase of prolonged microvascular retention and emigration is mediated by interactions between cognate adhesion molecules on the PMN and endothelium (1).

Concurrent with the events leading to the microvascular sequestration, PMN become activated, which is essential for subsequent emigration from the vascular space and ensuing microbicidal responses (1). Until recently, this activation has been assumed to be mediated predominantly by two mechanisms: interaction of soluble or surface-bound factors such as complement fragments (C5a), cytokines (interleukin-8), lipid mediators (leukotriene B₄, platelet-activating factor), and bacterial products (lipopolysaccharide, formyl peptides) with receptors on the plasma membrane of PMN and by "outside-in" signaling by adhesion receptors, including L-selectin (15) and CD11/CD18 (13). Both of these mechanisms induce activation of diverse intracellular signaling pathways leading to a series of rapid and coordinated ("effector") responses designed to allow PMN to reach an area of inflammation, destroy invading microorganisms, and remove inflammatory debris. These responses include motility (requiring complex cytoskeletal reorganization), phagocytosis, secretion of proteolytic enzymes and bactericidal proteins, and production of ROI leading to microbial destruction (7). In situations of inflammatory tissue injury, these same microbicidal products injure host tissues.

The present report by Kitagawa et al. (11) expands our views on the events that occur during the crucial seconds of the passage of PMN through microvascular beds such as in the lung. It is important to recall that the pulmonary capillaries are the major site of the physiological marginated pool of PMN, which accounts for 75% of all intravascular PMN (10). These cells serve as a rapidly mobilizable pool of phagocytes in close proximity to an interface (the alveolar-capillary membrane) where the vascular space and the increasingly hostile external environment are juxtaposed. Thus the observation that the deformation imposed on the PMN by the geometric constraints of the capillaries serves to activate the cells is indeed of fundamental importance both from the perspective of host defense and for the potential for host injury. As discussed above, an increase in F-actin and enhanced surface expression of CD11b/CD18 induced by mechanical deformation would serve to potentiate the sequestration in any subsequent capillary segments encountered by the PMN (and note that there may be up to 100 more segments encountered by such a PMN during transit through the lung). A transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) could serve to initiate or potentiate diverse intracellular signaling pathways, including activation of protein kinase C (14), which is known to influence many downstream events. Secretion of granule contents can also be induced by an increase in [Ca²⁺]_i leading to release of potentially injurious compounds such as proteolytic enzymes and cationic proteins. Importantly, CD11b/CD18 is contained within secondary granules (3), which might explain the increased surface expression of this important adhesion molecule induced by mechanical deformation.

Taking the above discussion at face value, it may seem surprising that PMN are not in a constant state of activation. However, common sense dictates that this cannot possibly be the case and leads us to the conclusion that counterregulatory mechanisms must exist to prevent uncontrolled activation of these potentially destructive cells. One such factor is the hydrodynamic force exerted by continued blood flow that tends to propel these cells through the microvascular bed. Anti-inflammatory factors released by the endothelium such as prostaglandin I₂, which leads to an increase in intracellular adenosine 3,5-cyclic monophosphate, could also attenuate leukocyte activation including actin polymerization (6), release of granule contents, and the oxidative burst (17). Undoubtedly, other mechanisms exist that prevent or attenuate activation of PMN by mechanical deformation.

As for any important observation, the results of the present study spawn additional questions and suggest additional avenues for investigation. Some of the questions that immediately spring to mind include the following. What is responsible for sensing the mechanical deformation? A likely candidate would be the cytoskeleton (composed of actin and tubulin), and a logical set of experiments would include pretreating the cells with microfilament (e.g., cytochalasins)- or microtubule (e.g., nocodozale)- disrupting agents and observing the effects on Ca²⁺ transients and CD11/CD18 expression. Alterations in membrane symmetry induced by mechanical deformation would be another potential sensing mechanism. Flow-cytometric analysis using fluorescent dyes sensitive to membrane symmetry could provide some insight into this possibility. Engagement of adhesion receptors on the PMN by the albumin coating the filters could be another mechanism of cell activation. If so, pretreatment of the cells with blocking antibodies against ₂-integrins would be expected to prevent the cytoskeletal changes and Ca²⁺ flux. Does mechanical deformation result in activation of ₂-integrins? The use of antibodies specific for the activated conformation of the ₂-integrin could be used to answer this question. Alternately, adhesion of filtered cells to and transmigration through endothelial monolayers could be measured as a functional assay for integrin activation. Can other types of mechanical forces induce similar changes? The use of magnetic twisting cytometry would be a logical starting point using ligands that bind to receptors linked to the cytoskeleton such as the ₂-integrins. Would the presence of endothelial cells modify the activation of leukocytes in transit through capillaries? Such experiments would require a technical tour de force but would bring these observations one step closer to the situation in vivo. Finally, what other signaling pathways are activated by mechanical deformation? Logical candidates to be studied would include protein kinase C, members of the mitogen-activated protein kinase family (p42-44 Erks, p38, c-Jun NH₂-terminal kinase), p21-activated kinase, and the renaturable kinases.

In summary, the observations by Kitagawa et al. (11) have opened our eyes to an important facet of mechanotransduction in leukocytes that has previously gone unrecognized. The notion that leukocytes can be activated by mechanical deformation imposed by geometric constraints of the capillaries has important implications for our understanding of the behavior of these cells in both physiological and pathological circumstances.

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