Summary

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Published: September 22, 2020
doi:

Summary

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Abstract

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

Introduction

We describe here efficient and reliable methods of differentiating intravascular leukocytes from pulmonary leukocytes. Even with the best perfusion techniques, studies have revealed residual CD45+ from circulation persists in the lung. This impairs the ability to distinguish between the rhythms in the circulation and the lung. This effect is further amplified in cases of lung inflammation. This is particularly relevant for the study of circadian regulation of inflammation.

Circadian rhythms refer to the diurnal oscillations in various biological processes that occur with a period of 24 h. The circadian system is an evolutionarily conserved anticipatory mechanism that confers protection on the host as it faces changes in its environment such as threat of infections. At the cellular level, the clock is organized into self-sustained transcriptional-translational feedback loops comprising the core clock genes1. The immune system has its own clock that impacts its response to pathogens and inflammatory insults2,3. As an organ exposed to the environment constantly, circadian rhythms are particularly important in the lung4. Various immune processes in the lung are under clock control5,6,7. However, the phase of various biological processes in the lung and the systemic circulation are not the same8, which by extension, also suggests that the oscillations of leukocytes in the lung and the circulation may not be identical. Thus, having a method to efficiently distinguish between pulmonary and intravascular leukocytes will be critical in the circadian context.

The aim of this study was to devise a method that can differentiate between intravascular and intraparenchymal leukocytes reliably. For this, we used a labeling of intravascular leukocytes and lung digestion method. For the labeling of intravascular leukocytes, we use intrajugular injection, which targets a large blood vessel and can be reproducibly used in mice of all strains and sizes. Many other methods have used tail vein injection9,10, which are notoriously harder to perform in Bl6 mice11. The intrajugular injection does necessitate use of anesthesia and is best done under direct visualization with dissecting microscope or magnifying loupes. Thus, the ease and reliability of the intrajugular injection should be weighed against the need for anesthesia and special equipment. However, given the ready availability of these equipment in most research labs, we do not view this to be a limiting factor. However, a case-by-case consideration seems prudent.

Protocol

All animal studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee and met the stipulations of the Guide for the Care and Use of Laboratory Animals. NOTE: The overall process may be divided into 1) intravenous CD45 labeling, 2) harvest, 3) digestion, and 4) staining and flow cytometry. These steps have been summarized in Figure 1. 1. Solutions/Reagent preparation Prepare Dissociati…

Representative Results

Using this technique, the total cell count of the naïve dissociated lungs (only the left lobes were used for the representative data) was between 27.3 x 106 to 71.1 x 106 cells/mL. After gating on size and gating out doublets and dead cells (gating scheme in Figure 2), the leukocyte counts ranged from 6.9 x 106 to 13.5 x 106 cells/mL. Circulating leukocytes that remain trapped even after perfusion to clear the lungs constituted approximately 4…

Discussion

Careful studies of lung inflammation and pulmonary immune responses are crucial to the understanding of many disease conditions. Flow cytometry is routinely used to enumerate and ascribe functional relevance to pulmonary leukocytes. The function of leukocytes depends at least partly on where they are found. Although there is accumulating evidence to support that even after perfect perfusion protocols, many intravascular leukocytes persist in the lungs, most studies do not differentiate between intrapulmonary and intravas…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the NHLBI-K08HL132053 (SS). The authors thank Dr. G. A. FitzGerald for access to a dissecting microscope and a shaking water bath.

Materials

Boekel Scientific Medium Water Bath Boekel Grant Scientific 290200
10 mL BD Syringes with BD Luer-Lok Tip BD Biosciences 309604
5 mL BD Syringes with BD Luer-Lok Tip BD Biosciences 309646
Anti-CD45- Pac Blue Biolegend 103114
Anti-CD45- Pe/Cy7 Biolegend 103114
Cell strainer 70 µm Nylon Fisher 352350
Corning Conical-Bottom Centrifuge Tube 50 mL Avantor 21008-714
Corning Falcon Test Tube with Cell Strainer Snap Cap EMSCO 10004637
Dissection Microscope Olympus SZX-SDO2
DMEM, high glucose Life Technologies 11965084
Dnase Roche 10104159001
DPBS without Ca++ & Mg++ 14190136
Fc Block Biolegend 101320
HyClone Fetal Bovine Serum GE Healthcare SH30071.03
L-Glutamine (200 mM) Life Technologies 25030-081
Liberase Research Grade Sigma 5401127001
Penicillin-Streptomycin (10,000 U/mL) Life Technologies 15140-122
Precision Shaking Water Bath Thermo Fisher TSSWB15
Red Blood Cell Lysing Buffer Sigma R7757
Suture Silk 4-0 Roboz SUT-15-2

References

  1. Partch, C. L., Green, C. B., Takahashi, J. S. Molecular architecture of the mammalian circadian clock. Trends in Cell Biology. 24, 90-99 (2014).
  2. Man, K., Loudon, A., Chawla, A. Immunity around the clock. Science. 354, 999-1003 (2016).
  3. Haspel, J. A., et al. Perfect timing: circadian rhythms, sleep, and immunity – an NIH workshop summary. JCI Insight. 5, (2020).
  4. Nosal, C., Ehlers, A., Haspel, J. A. Why Lungs Keep Time: Circadian Rhythms and Lung Immunity. Annual Review of Physiology. 82, 391-412 (2020).
  5. Gibbs, J., et al. An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action. Nature Medicine. 20, 919-926 (2014).
  6. Ehlers, A., et al. BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes. Mucosal Immunology. 11, 97-111 (2018).
  7. Sengupta, S., et al. Circadian control of lung inflammation in influenza infection. Nature Communications. 10, 4107 (2019).
  8. Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E., Hogenesch, J. B. A circadian gene expression atlas in mammals: implications for biology and medicine. Proceedings of the National Academy of Sciences of the United States of America. 111, 16219-16224 (2014).
  9. Anderson, K. G., et al. Intravascular staining for discrimination of vascular and tissue leukocytes. Nature Protocols. 9, 209-222 (2014).
  10. Gibbings, S. L., Jakubzick, C. V. Isolation and Characterization of Mononuclear Phagocytes in the Mouse Lung and Lymph Nodes. Methods in Molecular Biology. 1809, 33-44 (2018).
  11. Vines, D. C., Green, D. E., Kudo, G., Keller, H. Evaluation of mouse tail-vein injections both qualitatively and quantitatively on small-animal PET tail scans. Journal of Nuclear Medicine Technology. 39, 264-270 (2011).
  12. Tighe, R. M., et al. Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report. American Journal of Respiratory Cell and Molecular Biology. 61, 150-161 (2019).
  13. Anderson, K. G., et al. Cutting edge: intravascular staining redefines lung CD8 T cell responses. Journal of Immunology. 189, 2702-2706 (2012).
  14. Gibbings, S. L., et al. Three Unique Interstitial Macrophages in the Murine Lung at Steady State. American Journal of Respiratory Cell and Molecular Biology. 57, 66-76 (2017).
  15. Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model. Lab Animals (NY). 37, 26-32 (2008).
  16. Ho, D., et al. Heart Rate and Electrocardiography Monitoring in Mice. Current Protocols in Mouse Biology. 1, 123-139 (2011).
This article has been published
Video Coming Soon
Keep me updated:

.

Cite This Article
Issah, Y., Naik, A., Tang, S. Y., Forrest, K., Theken, K. N., Sengupta, S. Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach. J. Vis. Exp. (163), e61590, doi:10.3791/61590 (2020).

View Video