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Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus

Nature Medicine volume 19, pages 107–112 (2013)Cite this article

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A Corrigendum to this article was published on 06 April 2016

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Abstract

In contrast with many capillary beds, the glomerulus readily supports leukocyte recruitment. However, little is known regarding the actions of leukocytes following their recruitment to glomeruli. We used multiphoton confocal microscopy to examine leukocyte behavior in the glomerular microvasculature. In normal glomeruli, neutrophils and monocytes were retained in capillaries for several minutes, remaining static or migrating intravascularly. Induction of glomerular inflammation resulted in an increase in the duration of retention of static and migratory leukocytes. In response to immune complex deposition, both static and migratory neutrophils generated oxidants in inflamed glomeruli via a Mac-1–dependent mechanism. Our results describe a new paradigm for glomerular inflammation, suggesting that the major effect of acute inflammation is to increase the duration of leukocyte retention in the glomerulus. Moreover, these findings describe a previously unknown form of multicellular intravascular patrolling that involves both monocytes and neutrophils, which may underlie the susceptibility of the glomerulus to inflammation.

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Figure 1: Multiphoton microscopy reveals retention and migration of neutrophils and monocytes in the un-inflamed glomerular microvasculature.
Figure 2: Inflammation increases the duration of neutrophil retention and migration in glomerular capillaries.
Figure 3: Neutrophils and monocytes undergo retention and intraglomerular migration in intact (non-hydronephrotic) kidneys.
Figure 4: Nox2-dependent oxidant production is detectable in static and crawling neutrophils, and mediates glomerular injury.
Figure 5: Mac-1 mediates glomerular neutrophil recruitment and renal injury induced by GBM antibody.

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  • 12 August 2015

    In the published article, in the Online Methods section, it is stated that the dose of DHE used is 20 mg/kg, when in fact DHE was administered at 2 mg/kg. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors gratefully acknowledge the assistance of J. Peti-Peterdi for advice on intravital imaging of the kidney, T. Graf (Center for Genomic Regulation) for LysM-eGFP mice, M. Tymms and E. Williams for assistance with fluorochrome labeling, D.-B. Borza (Vanderbilt University School of Medicine) for providing 8D1 monoclonal antibody, and C. Lo for technical assistance. This work was supported by a National Health and Medical Research Council (Australia) Program grant (#334067), a National Health and Medical Research Council (Australia) Project grant (#606564) and a Genzyme Renal Innovations Program Project grant. M.J.H. is a National Health and Medical Research Council (Australia) Senior Research Fellow.

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Authors and Affiliations

  1. Monash University Department of Medicine, Centre for Inflammatory Diseases, Monash Medical Centre, Clayton, Victoria, Australia

    Sapna Devi, Anqi Li, Clare L V Westhorpe, Latasha D Abeynaike, Sarah L Snelgrove, Pam Hall, Joshua D Ooi, A Richard Kitching & Michael J Hickey

  2. Monash Micro Imaging, Monash University, Clayton, Victoria, Australia

    Camden Y Lo

  3. Department of Pharmacology, Monash University, Clayton, Victoria, Australia

    Christopher G Sobey

  4. Departments of Nephrology and Pediatric Nephrology, Monash Medical Centre, Clayton, Victoria, Australia

    A Richard Kitching

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Contributions

S.D. designed and performed the experiments and wrote the paper. A.L., C.L.V.W., L.D.A., S.L.S., P.H. and J.D.O. performed the experiments. C.Y.L. and C.G.S. contributed analytic tools. A.R.K. and M.J.H. designed the study and wrote the paper.

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Correspondence to Michael J Hickey.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 375 kb)

Supplementary Video 1

Neutrophil adhesion and migration in an uninflamed glomerulus. Video of a glomerulus in a hydronephrotic kidney of an untreated wild-type mouse. Gr-1+ neutrophils (red) can be seen undergoing periods of transient retention and migration within glomerular capillaries (glomerular capillaries stained with Alexa 488-conjugated isolectin GS-IB4). (Duration in real time = 10 min). (MOV 345 kb)

Supplementary Video 2

Retention and migration of GFP+ leukocytes in an uninflamed glomerulus of a CX3CR1GFP/+ mouse. Video of a glomerulus in a hydronephrotic kidney of an untreated CX3CR1GFP/+ mouse. A GFP+ monocyte can be seen undergoing prolonged migration in the glomerulus (capillaries labelled with rhodamine dextran) in the absence of an inflammatory stimulus. (Duration in real time = 35 min). (MOV 150 kb)

Supplementary Video 3

Neutrophil adhesion and migration in a control (NSG-treated) mouse. Video of a glomerulus of a wild-type mouse in the first hour after receiving non-immune sheep globulin (NSG) as control for anti-GBM Ab. Gr-1+ neutrophils (red) can be seen undergoing periods of transient retention and migration within glomerular capillaries (glomerular capillaries stained with Alexa 488-conjugated isolectin GS-IB4), consistent with the glomerular leukocyte behaviour seen in untreated mice. (Duration in real time = 20 min 30 s). (AVI 3742 kb)

Supplementary Video 4

Neutrophil adhesion and migration in an inflamed glomerulus. Video of a glomerulus of a wild-type mouse in the first hour following administration of anti-GBM Ab. Gr-1+ neutrophils (red) can be seen undergoing sustained interactions within glomerular capillaries (glomerular capillaries stained with Alexa 488-conjugated isolectin GS-IB4), either remaining static, or migrating within glomerular capillaries. (Duration in real time = 25 min). (AVI 4412 kb)

Supplementary Video 5

Leukocyte retention in capillaries of inflamed skeletal muscle. Multiphoton imaging of the cremaster muscle microvasculature following local TNF injection. Gr-1+ neutrophils (red) are visible moving within muscle capillaries (stained with FITC-conjugated isolectin GS-IB4). (Duration in real time = 10 min). (AVI 4437 kb)

Supplementary Video 6

Neutrophil adhesion and migration in an uninflamed glomerulus of an intact kidney. Video of a glomerulus in an intact (non-hydronephrotic) kidney of a wild-type mouse treated with NSG. Gr-1+ neutrophils (green) are visible undergoing periods of transient retention and migration within glomerular capillaries (vasculature labelled with rhodamine dextran). (Duration in real time = 20 min). (AVI 4217 kb)

Supplementary Video 7

Neutrophil adhesion and migration in an inflamed glomerulus of an intact kidney. Video of a glomerulus in an intact kidney of a wild-type mouse treated with anti-GBM Ab. Gr-1+ neutrophils (green) are visible undergoing lengthy periods of retention and migration within glomerular capillaries (vasculature labelled with rhodamine dextran). (Duration in real time = 20 min). (AVI 3883 kb)

Supplementary Video 8

Monocyte migration in an uninflamed glomerulus of an intact kidney. Video of a glomerulus in an intact kidney of an untreated CX3CR1GFP/+ mouse. A GFP+ monocyte can be seen undergoing prolonged migration in the glomerulus (capillaries labelled with rhodamine dextran). (Duration in real time = 11 min 30 s). (MOV 229 kb)

Supplementary Video 9

Neutrophil oxidant generation in uninflamed glomeruli detected by DHE. Video shows a glomerulus of a wild-type mouse 1–2 hrs after receiving NSG, and DHE (to facilitate detection of oxidant generation). Gr-1+ neutrophils (green) can be seen undergoing periods of transient retention and migration within glomerular capillaries (labelled with Pacific blue dextran), while minimal DHE-associated fluorescence (red) is detectable. Autofluorescent cells are present adjacent to the glomerulus. (Duration in real time = 5 min). (MOV 274 kb)

Supplementary Video 10

Neutrophil oxidant generation in inflamed glomeruli detected by DHE. Video shows a glomerulus of a wild-type mouse 1–2 hrs after receiving anti-GBM Ab, and DHE (to facilitate detection of oxidant generation). Gr-1+ neutrophils (green) can be seen undergoing sustained interactions within glomerular capillaries (labelled with Pacific blue dextran). Some neutrophils also display intracellular DHE-associated fluorescence (red). DHE+ neutrophils either remain static, or migrate within glomerular capillaries. Autofluorescent cells are present adjacent to the glomerulus. (Duration in real time = 5 min). (MOV 281 kb)

Supplementary Video 11

Neutrophil oxidant generation in Nox2-deficient mouse. Video shows a glomerulus of a Cybb−/− mouse 1–2 hrs after receiving anti-GBM Ab, and DHE, to facilitate detection of oxidant generation. Gr-1+ neutrophils (green) can be seen undergoing sustained static or migratory interactions within glomerular capillaries (labelled with Pacific blue dextran). However, no neutrophils show detectable DHE-associated fluorescence. (Duration in real time = 20 min). (MOV 907 kb)

Supplementary Video 12

Neutrophil behaviour in glomeruli of anti-GBM Ab-treated mouse following Mac-1 inhibition. Video of a glomerulus in an anti-GBM Ab-treated wild-type mouse following pretreatment with anti-Mac-1. 1–2 hrs after administration of anti-GBM Ab, interactions of Gr-1+ neutrophils in glomerular capillaries are predominantly transient, with cells subsequently exiting the glomerulus. (Duration in real time = 20 min). (AVI 3426 kb)

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Devi, S., Li, A., Westhorpe, C. et al. Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus. Nat Med 19, 107–112 (2013). https://doi.org/10.1038/nm.3024

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