Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.

[1]  G. Whitesides,et al.  Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device , 2002, Nature Biotechnology.

[2]  L. Cancio,et al.  Evaluation of white blood cell count, neutrophil percentage, and elevated temperature as predictors of bloodstream infection in burn patients. , 2007, Archives of surgery.

[3]  D. Beebe,et al.  Selective and tunable gradient device for cell culture and chemotaxis study. , 2009, Lab on a chip.

[4]  O. Laerum,et al.  Altered neutrophil functions in patients with large burns. , 1990, Blood cells.

[5]  J. Alexander,et al.  Flow cytometric analysis of neutrophil subsets in thermally injured patients developing infection. , 1990, Clinical immunology and immunopathology.

[6]  M. Toner,et al.  Rapid Appearance of Resolvin Precursors in Inflammatory Exudates: Novel Mechanisms in Resolution1 , 2008, The Journal of Immunology.

[7]  Mehmet Toner,et al.  Neutrophil migration assay from a drop of blood. , 2008, Lab on a chip.

[8]  Jose Salinas,et al.  Closed-loop and decision-assist resuscitation of burn patients. , 2008, The Journal of trauma.

[9]  C. White,et al.  Advances in surgical care: Management of severe burn injury , 2008, Critical care medicine.

[10]  J. Solomkin Neutrophil disorders in burn injury: complement, cytokines, and organ injury. , 1990, The Journal of trauma.

[11]  T. Coates,et al.  The fundamental motor of the human neutrophil is not random: evidence for local non-Markov movement in neutrophils. , 1994, Biophysical journal.

[12]  Marcus Spies,et al.  Effects of delayed wound excision and grafting in severely burned children. , 2002, Archives of surgery.

[13]  J. Wikswo,et al.  Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator. , 2005, Lab on a chip.

[14]  R. Nelson,et al.  Thermal injury, the inflammatory process, and wound dressing reduce human neutrophil chemotaxis to four attractants. , 1993, The Journal of burn care & rehabilitation.

[15]  J. Solomkin,et al.  Regulation of Neutrophil Migratory Function in Burn Injury by Complement Activation Products , 1984, Annals of surgery.

[16]  G. Charras,et al.  Polar stimulation and constrained cell migration in microfluidic channels. , 2007, Lab on a chip.

[17]  S. Boyden THE CHEMOTACTIC EFFECT OF MIXTURES OF ANTIBODY AND ANTIGEN ON POLYMORPHONUCLEAR LEUCOCYTES , 1962, The Journal of experimental medicine.

[18]  John M. Adams,et al.  Early trauma polymorphonuclear neutrophil responses to chemokines are associated with development of sepsis, pneumonia, and organ failure. , 2001, The Journal of trauma.

[19]  G. O’Keefe,et al.  An evaluation of risk factors for mortality after burn trauma and the identification of gender-dependent differences in outcomes. , 2001, Journal of the American College of Surgeons.

[20]  W. Lippert,et al.  Polymorphonuclear leucocyte motility in patients with severe burns. , 1989, Burns : journal of the International Society for Burn Injuries.

[21]  Albert Folch,et al.  A new method for studying gradient-induced neutrophil desensitization based on an open microfluidic chamber. , 2010, Lab on a chip.

[22]  R. Gamelli,et al.  American Burn Association Consensus Conference to Define Sepsis and Infection in Burns , 2007, Journal of burn care & research : official publication of the American Burn Association.

[23]  A. Mason,et al.  Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. , 1974, The Journal of clinical investigation.

[24]  S. Malawista,et al.  Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Alterations in leukocyte adhesion molecule expression after burn injury. , 1995, The Journal of trauma.

[26]  Zigmond Sh Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. , 1977 .

[27]  Mehmet Toner,et al.  Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients. , 2006, Lab on a chip.

[28]  D. Fearon,et al.  Neutrophil activation in thermal injury as assessed by increased expression of complement receptors. , 1986, The New England journal of medicine.

[29]  Allogeneic blood transfusion increases the risk of postoperative bacterial infection: a meta-analysis. , 2003 .

[30]  R. Simmons,et al.  Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. , 1975, Journal of immunology.

[31]  Shur-Jen Wang,et al.  Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration. , 2004, Biochemical and biophysical research communications.

[32]  P. Rhee,et al.  New developments in fluid resuscitation. , 2007, The Surgical clinics of North America.

[33]  S. Zigmond,et al.  ABILITY OF POLYMORPHONUCLEAR LEUKOCYTES TO ORIENT IN GRADIENTS OF CHEMOTACTIC FACTORS , 2003 .

[34]  H U Keller,et al.  Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. , 1981, Journal of cell science.

[35]  E. Deitch,et al.  Effect of local and systemic burn microenvironment on neutrophil activation as assessed by complement receptor expression and morphology. , 1990, The Journal of trauma.

[36]  J. Saffle The Phenomenon of “Fluid Creep” in Acute Burn Resuscitation , 2007, Journal of burn care & research : official publication of the American Burn Association.

[37]  D. Schoenfeld,et al.  Significant Reductions in Mortality for Children With Burn Injuries Through the Use of Prompt Eschar Excision , 1988, Annals of surgery.

[38]  A. Bjornson,et al.  Down-regulation of chemotaxis of polymorphonuclear leukocytes following thermal injury involves two distinct mechanisms. , 1993, The Journal of infectious diseases.

[39]  P. Lipsky,et al.  Interference with the Function of Leukocyte Adhesion Molecules by Monoclonal Antibodies: A New Approach to Burn Injury , 1994, European journal of pediatric surgery : official journal of Austrian Association of Pediatric Surgery ... [et al] = Zeitschrift fur Kinderchirurgie.

[40]  David J Beebe,et al.  Characterization of a membrane-based gradient generator for use in cell-signaling studies. , 2006, Lab on a chip.

[41]  D. Gerneke,et al.  Cytoskeletal actin: the influence of major burns on neutrophil structure and function. , 1994, Burns : journal of the International Society for Burn Injuries.

[42]  B. Chung,et al.  Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber , 2007, Biomedical microdevices.

[43]  Mehmet Toner,et al.  A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects. , 2007, Lab on a chip.

[44]  B. Chung,et al.  A microfluidic multi-injector for gradient generation. , 2006, Lab on a chip.

[45]  A. Oudenaarden,et al.  Neutrophil chemorepulsion in defined interleukin‐8 gradients in vitro and in vivo , 2006, Journal of leukocyte biology.

[46]  E. Lightfoot,et al.  Inhibition of leukocyte adherence in a rabbit model of major thermal injury. , 1993, The Journal of burn care & rehabilitation.

[47]  E. Lightfoot,et al.  Inhibition of leukocyte adherence and susceptibility to infection. , 1993, The Journal of surgical research.

[48]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[49]  D Zicha,et al.  A new direct-viewing chemotaxis chamber. , 1991, Journal of cell science.

[50]  Colin Song,et al.  Meta-analysis of early excision of burns. , 2006, Burns : journal of the International Society for Burn Injuries.

[51]  G. Arturson,et al.  Neutrophil leucocyte functions and wound bacteria in burn patients , 1977 .

[52]  D. Herndon,et al.  Post burn muscle wasting and the effects of treatments. , 2005, The international journal of biochemistry & cell biology.

[53]  J. Wikswo,et al.  Microfluidic switching system for analyzing chemotaxis responses of wortmannin-inhibited HL-60 cells , 2008, Biomedical microdevices.

[54]  Mehmet Toner,et al.  Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients. , 2008, Clinical chemistry.

[55]  R. Barrow,et al.  A Comparison of Conservative Versus Early Excision: Therapies in Severely Burned Patients , 1989, Annals of surgery.

[56]  David J Beebe,et al.  A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment. , 2008, Lab on a chip.

[57]  Francis Lin,et al.  Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices , 2005, Annals of Biomedical Engineering.

[58]  Mingming Wu,et al.  A hydrogel-based microfluidic device for the studies of directed cell migration. , 2007, Lab on a chip.

[59]  D. Herndon,et al.  The pharmacologic modulation of the hypermetabolic response to burns. , 2005, Advances in surgery.

[60]  Albert Folch,et al.  Measurement of cell migration in response to an evolving radial chemokine gradient triggered by a microvalve. , 2006, Lab on a chip.

[61]  D. Chinkes,et al.  Randomized Controlled Trial to Determine the Efficacy of Long-Term Growth Hormone Treatment in Severely Burned Children , 2009, Annals of surgery.

[62]  L. Cancio,et al.  Contribution of bacterial and viral infections to attributable mortality in patients with severe burns: an autopsy series. , 2010, Burns : journal of the International Society for Burn Injuries.

[63]  S. Arbabi,et al.  Advances in burn critical care , 2006, Critical care medicine.

[64]  A. Meyer,et al.  Lack of correlation between decreased chemotaxis and susceptibility to infection in burned rats. , 1986, The Journal of trauma.

[65]  A. Finn,et al.  Neutrophil disorders and their management , 2001, Journal of clinical pathology.

[66]  A. Mason,et al.  Suppression of Leukocyte Chemotaxis in vitro by Chemotherapeutic Agents Used in the Management of Thermal Injuries , 1975, Annals of surgery.

[67]  D. Chinkes,et al.  The leading causes of death after burn injury in a single pediatric burn center , 2009, Critical care.