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A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients

Nature Medicine volume 7, pages 376–379 (2001)Cite this article

Inflammatory lesions, regardless of whether they are caused by ischemia/reperfusion, trauma, infections or immunological stimuli, are characterized by a local accumulation of leukocytes1,2. The extravasation of leukocytes from the blood vessels into inflamed tissues is initiated by a complex cascade of molecular interactions between the selectin family and their sialyl Lewis X-containing ligands, leading to rolling of leukocytes3,4,5. We describe here the use of a novel non-invasive, in vivo technique based on reflected-light, tandem-scanning confocal microscopy of capillaries to observe leukocyte rolling during lesional inflammation in human patients. A well-characterized inflammation was induced by elective cataract surgery. We observed very few rolling or tissue-infiltrating leukocytes in preoperative analysis. However, at the sites of inflammation, we found a 40-fold increase in the number of rolling cells, with a concomitant significant decrease in leukocyte velocity. At the same time, the number of tissue-infiltrating cells was significantly elevated compared with control values. As the clinical inflammation resolved, the number of rolling and extravasated leukocytes decreased back to normal levels. Our data indicate that this novel in vivo technique, which can be applied to human patients repeatedly, will allow the quantitative evaluation of inflammatory conditions or their therapeutic intervention—a task that has previously only been approached invasively.

Leukocyte rolling has been extensively studied with intravital microscopy in various experimental inflammatory models in animals6,7,8, but not yet in humans. The rolling and adhesion phases are important but do not predict the actual extravasation of leukocytes into tissues, and thus other approaches are needed for the analysis of inflammation9. In animal studies, cells that have migrated into the tissue have been quantified, for example, using histology from tissue biopsies, local accumulation of leukocyte-specific myeloperoxidase enzyme activity from the tissue homogenates or in vivo recruitment of radiolabeled leukocytes into tissues1,10,11. As none of these methods is easily applicable for repeated analysis in human patients, we used reflected-light confocal microscopy in vivo for the non-invasive analysis of leukocyte rolling on capillary endothelium at sites of inflammation. In contrast to traditional confocal microscopes with only one pinhole or slit for emission and detection, our microscope has a rotating Nipkow disk with thousands of pinholes, allowing real-time analysis of rapidly moving particles12,13. Another important feature is the use of reflected light combined with the tandem-scanning principle, which provides bright illumination of widely scattering particles (for example, leukocytes in blood and tissue) in a 9-μm focal plane. This microscopic technique does not cause trauma and has been approved by the United States Food and Drug Administration for use in the examination of the human eye14.

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Figure 1: In vivo analysis of leukocyte rolling in human capillaries.
Figure 2: In vivo analysis of tissue-emigrated leukocytes at the site of inflammation in human conjunctiva.

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Acknowledgements

We thank M. Salmi and S. Tohka for discussions on leukocyte rolling and S. Sarna for help with statistical analysis. This work was supported by grants from the Academy of Finland 101-36157 (R.R.), Technology Development Center of Finland 40016/99 (R.R.), the Emil Aaltonen Foundation (R.R.), the Rector's grant, Helsinki University (T.T.), research funds from the Health Care Region of Helsinki and Uusimaa (T.T. and R.R.), Mary and Georg C. Ehrnrooth Foundation (MHV) and the Eye Foundation (JAOM).

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Authors and Affiliations

  1. Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland

    Juha Kirveskari & Risto Renkonen

  2. HUCH Laboratory Diagnostics, the Health Care Region of Helsinki and Uusimaa, Finland

    Juha Kirveskari & Risto Renkonen

  3. Department of Ophthalmology, University of Helsinki, Helsinki, Finland

    Minna H. Vesaluoma, Jukka A.O. Moilanen, Timo M.T. Tervo & Eeva Linnolahti

  4. Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Matthew W. Petroll

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  1. Juha Kirveskari
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Correspondence to Risto Renkonen.

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Kirveskari, J., Vesaluoma, M., Moilanen, J. et al. A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients. Nat Med 7, 376–379 (2001). https://doi.org/10.1038/85538

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