Skip to main content
SpringerLink
Log in

Collection of In Vivo Transmigrated Neutrophils from Human Skin

  • Protocol
  • First Online:
Neutrophil Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1124))

Abstract

A wealth of knowledge on the life and death of human neutrophils has been obtained by the in vitro study of isolated cells derived from peripheral blood. However, neutrophils are of main importance, physiologically as well as pathologically, after they have left circulation and transmigrated to extravascular tissues. The journey from blood to tissue is complex and eventful, and tissue neutrophils are in many aspects distinct from the cells left in circulation. Here we describe how to obtain human tissue neutrophils in a controlled experimental setting from aseptic skin lesions created by the application of negative pressure. One protocol enables the direct analysis of the blister content, infiltrating leukocytes as well as exudate fluid, and is a simple method to follow multiple parameters of aseptic inflammation in vivo. Also described is the skin chamber technique, a method based on denuded skin blisters which are subsequently covered by collection chambers filled with autologous serum. Although slightly more artificial as compared to analysis of the blister content directly, the cellular yield of this skin chamber method is sufficient to perform a large number of functional analyses of in vivo transmigrated cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Condliffe AM, Kitchen E, Chilvers ER (1998) Neutrophil priming: pathophysiological consequences and underlying mechanisms. Clin Sci (Lond) 94:461–471

    CAS  Google Scholar 

  2. Zarember KA, Kuhns DB (2011) Editorial: will the real neutrophil please stand up? J Leukocyte Biol 90:1039–1041

    Article  CAS  PubMed  Google Scholar 

  3. Kuhns DB, Long Priel DA, Gallin JI (1995) Loss of L-selectin (CD62L) on human neutrophils following exudation in vivo. Cell Immunol 164:306–310

    Article  CAS  PubMed  Google Scholar 

  4. Lukac J, Mravak-Stipetic M, Knezevic M et al (2003) Phagocytic functions of salivary neutrophils in oral mucous membrane diseases. J Oral Pathol Med 32:271–274

    Article  CAS  PubMed  Google Scholar 

  5. Briheim G, Stendahl O, Dahlgren C (1986) Factor-specific deactivation of leucocyte chemotaxis in vivo. Scand J Haematol 37:253–258

    Article  CAS  PubMed  Google Scholar 

  6. Hustinx WN, Van Kessel CP, Heezius E et al (1998) Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia. Clin Exp Immunol 112:334–340

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Christenson K, Björkman L, Karlsson A et al (2013) Regulation of neutrophil apoptosis differs after in vivo transmigration to skin chambers and synovial fluid: a role for inflammasome dependent IL-1β release. J Innate Immun 5:377–388

    Google Scholar 

  8. Kiistala U, Mustakallio KK (1964) In-vivo separation of epidermis by production of suction blisters. Lancet 1:1444–1445

    Article  CAS  PubMed  Google Scholar 

  9. Bork K (1978) Physical forces in blister formation. The role of colloid osmotic pressure and of total osmolality in fluid migration into the rising blister. J Investig Dermatol 71:209–212

    Article  CAS  PubMed  Google Scholar 

  10. Hunter JA, McVittie E, Comaish JS (1974) Light and electron microscopic studies of physical injury to the skin. I. Suction. Brit J Dermatol 90:481–490

    Article  CAS  Google Scholar 

  11. Kiistala U, Mustakallio KK (1967) Dermo-epidermal separation with suction. Electron microscopic and histochemical study of initial events of blistering on human skin. J Invest Dermatol 48:466–477

    CAS  PubMed  Google Scholar 

  12. Thorén FB, Riise RE, Ousbäck J et al (2012) Human NK Cells induce neutrophil apoptosis via an NKp46- and Fas-dependent mechanism. J Immunol 188:1668–1674

    Article  PubMed  Google Scholar 

  13. Follin P (1999) Skin chamber technique for study of in vivo exudated human neutrophils. J Immunol Methods 232:55–65

    Article  CAS  PubMed  Google Scholar 

  14. Christenson K, Björkman L, Karlsson J et al (2011) In vivo-transmigrated human neutrophils are resistant to antiapoptotic stimulation. J Leukoc Biol 90:1055–1063

    Article  CAS  PubMed  Google Scholar 

  15. Follin P, Briheim G, Dahlgren C (1991) Mechanisms in neutrophil priming: characterization of the oxidative response induced by formylmethionyl-leucyl-phenylalanine in human exudated cells. Scand J Immunol 34:317–322

    Article  CAS  PubMed  Google Scholar 

  16. Karlsson A, Follin P, Leffler H et al (1998) Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils. Blood 91:3430–3438

    CAS  PubMed  Google Scholar 

  17. Paulsson JM, Moshfegh A, Dadfar E et al (2012) In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils. Clin Exp Immunol 168:105–112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Sengelov H, Follin P, Kjeldsen L et al (1995) Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol 154:4157–4165

    CAS  PubMed  Google Scholar 

  19. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97:77–89

    CAS  PubMed  Google Scholar 

  20. Boyum A, Lovhaug D, Tresland L et al (1991) Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand J Immunol 34:697–712

    Article  CAS  PubMed  Google Scholar 

  21. Follin P, Wymann MP, Dewald B et al (1991) Human neutrophil migration into skin chambers is associated with production of NAP-1/IL8 and C5a. Eur J Haematol 47:71–76

    Article  CAS  PubMed  Google Scholar 

  22. Kuhns DB, DeCarlo E, Hawk DM et al (1992) Dynamics of the cellular and humoral components of the inflammatory response elicited in skin blisters in humans. J Clin Invest 89:1734–1740

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Day RM, Harbord M, Forbes A et al (2001) Cantharidin blisters: a technique for investigating leukocyte trafficking and cytokine production at sites of inflammation in humans. J Immunol Methods 257:213–220

    Article  CAS  PubMed  Google Scholar 

  24. Gowland E (1964) Studies on the emigration of polymorphonuclear leucocytes from skin lesions in man. J Pathol Bacteriol 87:347–352

    Article  CAS  PubMed  Google Scholar 

  25. Perillie PE, Finch SC (1964) Quantitative studies of the local exudative cellular reaction in acute leukemia. J Clin Invest 43:425–430

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Hellum KB, Solberg CO (1977) Human leucocyte migration: studies with an improved skin chamber technique. Acta Pathol Microbiol Scand C 85C:413–423

    CAS  PubMed  Google Scholar 

  27. Wright DG, Gallin JI (1979) Secretory responses of human neutrophils: exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J Immunol 123:285–294

    CAS  PubMed  Google Scholar 

  28. Kiistala U (1972) Dermal-epidermal separation. II. External factors in suction blister formation with special reference to the effect of temperature. Ann Clin Res 4:236–246

    CAS  PubMed  Google Scholar 

  29. Paulsson J, Dadfar E, Held C et al (2007) Activation of peripheral and in vivo transmigrated neutrophils in patients with stable coronary artery disease. Atherosclerosis 192:328–334

    Article  CAS  PubMed  Google Scholar 

  30. Thylen P, Lundahl J, Fernvik E et al (2000) Impaired monocyte CD11b expression in interstitial inflammation in hemodialysis patients. Kidney Int 57:2099–2106

    Article  CAS  PubMed  Google Scholar 

  31. Vignali DA (2000) Multiplexed particle-based flow cytometric assays. J Immunol Methods 243:243–255

    Article  CAS  PubMed  Google Scholar 

  32. Preijers FW, Huys E, Leenders M et al (2011) The new violet laser dye, Krome Orange, allows an optimal polychromatic immunophenotyping based on CD45-KO gating. J Immunol Methods 372:42–51

    Article  CAS  PubMed  Google Scholar 

  33. Theilgaard-Monch K, Knudsen S, Follin P et al (2004) The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing. J Immunol 172:7684–7693

    PubMed  Google Scholar 

  34. Martin TR, Raugi G, Merritt TL et al (1987) Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage. J Clin Invest 80:1114–1124

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Nigon F, Rouis M, Foster SJ et al (1991) Native low-density lipoproteins stimulate leukotriene B4 production by human monocyte-derived macrophages. Biochim Biophys Acta 1083:230–234

    Article  CAS  PubMed  Google Scholar 

  36. Grimm MC, Elsbury SK, Pavli P et al (1996) Interleukin 8: cells of origin in inflammatory bowel disease. Gut 38:90–98

    Article  CAS  PubMed  Google Scholar 

  37. Winkler AR, Nocka KH, Sulahian TH et al (2008) In vitro modeling of human alveolar macrophage smoke exposure: enhanced inflammation and impaired function. Exp Lung Res 34:599–629. PMID:23570945

    Google Scholar 

Download references

Acknowledgments

This work was supported by the Swedish Research Council (521-2009-3443), the King Gustav V Memorial Foundation, Gothenburg Medical Society, Ingabritt and Arne Lundgren’s Research Foundation, the Gothenburg Rheumatism Association, and the Swedish state under the LUA/ALF agreement.

Author information

Authors and Affiliations

  1. Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden

    Karin Christenson

  2. The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden

    Lena Björkman, Lisa Davidsson, Anna Karlsson, Claes Dahlgren & Johan Bylund

  3. Department of Communicable Disease Control and Prevention, Västra Götaland Region, Gothenburg, Sweden

    Per Follin

Authors
  1. Karin Christenson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lena Björkman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisa Davidsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Karlsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Per Follin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Claes Dahlgren
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Johan Bylund
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Montana State University Dept. Veterinary Molecular Biology, Bozeman, Montana, USA

    Mark T. Quinn

  2. Lab. Human Bacterial Pathogenesis, National Institutes of Health Nat. Inst. Allergy & Infectious Diseases, Hamilton, Montana, USA

    Frank R. DeLeo

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Christenson, K. et al. (2014). Collection of In Vivo Transmigrated Neutrophils from Human Skin. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-845-4_4

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-844-7

  • Online ISBN: 978-1-62703-845-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation