Characteristics and Applications of Human PB Neutrophils

Author: AcceGen R&D Team

What are Neutrophils?

The immune system is a series of complex defense responses in which the body performs immune responses and defends against the invasion of pathogens. Immunity from disease is divided into innate immunity (nonspecific immunity) and adaptive immunity (specific immunity). The nonspecific effector cells of the innate immune response include scavenger cells and natural killer cell. Among them, neutrophils are critical components of the body’s immune response to infection.

Based on the shape of the nucleus and the way granules in the cytoplasm are stained with dyes，granulocytes are divided into three categories – neutrophils, eosinophils, and basophils. Neutrophils, which ingest and destroy microorganisms, especially bacteria, are the most abundant type of granulocyte in the human body ^[2]. Granulocytes are mobile and are attracted by chemical signals to foreign materials. Sometimes the chemical signals are produced by the invading microorganism itself, others by damaged tissue, and some by the interaction between microbes and proteins in the blood plasma. Functions of neutrophil are modulated by the activating and inhibitory signaling pathways that are known to act downstream of ITAM- and ITIM-containing receptors. Moreover, the diverse range of immunoreceptors on neutrophils of a variety of different extracellular ligands regulate activating and inhibitory signaling. CD16, CD45, CD66b, etc. are cell surface markers commonly used in research.

(Figure 1. Tissue-specific recruitment of neutrophils ^[2])

Human Peripheral Blood Neutrophils or Human PB Neutrophils, have many applications, such as the study of migration and recruitment mechanisms; the occurrence and development of diseases (asthma, etc.), especially cancer; death mechanism research. Research on the association with new coronavirus pneumonia has also attracted much attention ^[1].

Culture Protocol of Human PB Neutrophils

Human pb Neutrophils provide an ideal cell culture model for studying its role in immune homeostasis, inflammation response and cancer biology.

Isolation of neutrophils using Percoll-based density gradient ^[3]

1 Spin Vacutainer tubes at 400 × g for 10 min.

2 Transfer plasma (upper phase) into new 15 ml conical tube and spin again to collect all leukocytes from plasma.

3 Remove platelet-rich plasma to new tube and spin for 10 min at ≥2000 × g to remove platelets.

4 Pour RBC-containing layer from Vacutainer tubes (up to 3) into 50 ml conical tube.

5 Pour HBSS(−) into the 50ml tube containing blood to the 25ml mark.

6 Add 25 ml of 3% dextran and invert 5 × to mix. Allow to sediment for 20–40 min.

7 Pipet the supernatant containing leukocytes into a fresh 50 ml conical tube leaving behind as much of the sedimented RBC layer as possible.

8 Top off tubes to 50 ml with HBSS(−) and centrifuge at 400 × g for 10 min.

9 Aspirate the supernatant and loosen the cell pellet by gently tapping tube.

10 Pipet 10 ml of 33mM NaCl into cells.

11 Within 30 seconds, add 10 ml 267mM NaCl to normalize osmolarity, then top off tube with HBSS(−).

12 Centrifuge for 10 min at 400 × g and aspirate the supernatant.

13 Repeat steps 9–12 again to reduce residual RBC.

14 Resuspend cell pellet containing leukocytes in 2 ml HBSS (−) by pipetting up and down.

15 Prepare a discontinuous Percoll gradient as follows:

Bottom layer: Mix 1.133ml 90% Percoll solution and 0.867 ml autologous plasma (51% Percoll)

Middle layer: Mix 0.933 ml 90% Percoll solution and 1.067ml autologous plasma (42% Percoll)

Layer drop-wise on bottom layer, leaving additional bubbles on top of layer and avoid moving tube.

Overlay 2ml resuspended leukocytes in HBSS(−) and centrifuge at 400 × g for 10 minutes.

16 PMN form a pellet at the bottom of the tube. Carefully aspirate upper layers leaving cell pellet at bottom. Resuspend PMN in 1 ml HBSS(−) by pipetting gently up and down. Transfer to fresh 15 ml conical tube containing 14 ml HBSS(−).

17 Centrifuge PMN as before and resuspend in 1–2 ml HBSS(−)

18 Count the cells using an automated cell counter. Alternatively, dilute 10 µl of cells in 490 µl of 3% acetic acid and count in a hemacytometer.

19 To assess purity of isolated PMN, dilute cells to between 5 – 10 × 105/ml in HBSS(−) and load 100 µl in a cytocentrifuge funnel (e.g., Cytospin 2, Shandon) following manufacturer’s instructions. Spin for 1 minute at 1,000 rpm to deposit a uniform layer of cells on the microscope slide. Differential stains (e.g., Diff-quick, or Hemacolor) should be used to accentuate differences between eosinophils, neutrophils, and other cell types.

Neutrophils in the Latest Research

In pathological conditions, neutrophils need to move to the site of infection to function, so studies on migration and recruitment mechanisms are important, which are driven by cytokine and chemokine signals directly targeting neutrophils via specific cell surface receptors. The researchers found that the IL-20 signaling pathway played an important role by detecting the expression of cytokines during simulated Staphylococcus aureus infection. Migration and activation of human neutrophils, including phagocytosis, granule exocytosis, and migration altered the expression of IL-20R chains, manifested as modification of actin polymerization and inhibition of a broad range of actin-dependent functions. This study suggested that during infection with S. aureus, IL-20 directly targets and inhibits neutrophils’ inflammatory functions ^[4].

Neutrophils have a short lifespan. Mature neutrophils must be rapidly cleared from inflammatory sites to prevent tissue damage. Neutrophil death can occur through multiple pathways and is regulated by interactions with commensal and pathogenic microorganisms, environmental exposures, as well as cellular age ^[5]. Neutrophil clearance and recruitment maintain a delicate balance, and once the balance is disrupted, it will directly affect circulating neutrophil numbers, cellular trafficking, antimicrobial defense, and host well-being. Depletion of neutrophils is thought to be accomplished primarily through caspase-dependent apoptosis. Studies have also shown that the clearance of human neutrophils may also be accomplished through autophagy. Some researchers found that neutrophils do not undergo DNA fragmentation, nor are they TUNEL positive, but express an autophagy marker, LC3-II. In addition, autophagy inhibitors prevented the division of the nucleus during differentiation, indicating that these cells underwent the process of autophagy ^[6].

Neutrophil granulocytes are critical mediators of innate immunity and tissue regeneration, potentially linking to many diseases, such as asthma. Bronchial asthma manifests as airway inflammation, airway hyperresponsiveness, reversible airway obstruction, and airway remodeling. Clearly, neutrophils are important players in development of asthma and play a role in asthma exacerbations ^[8]. Early in the disease, airway neutrophil chemotaxis increases and apoptosis decreases, resulting in an increase in airway neutrophil inflammation. Neutrophils can produce a variety of molecules, such as elastase, myeloperoxidase, chemokines and cytokines, which are involved in the occurrence and development of asthma. The impact of interfering with the neutrophilic component in asthma is a new research aspect in asthma treatment. More research is needed to fully explore how modulation of neutrophil activity can confer significant benefits to patients with asthma ^[7,8].

Neutrophils are a good entry point in cancer treatment and prognosis. In the treatment and prognosis of cancer, neutrophils are a good entry point. On the one hand, neutrophil-based drug delivery has gained increasing attention in preclinical models. In addition, different neutrophil components, conflicting functions of neutrophils, and activation of neutrophils are also important therapeutic targets for cancer therapy ^[9]. Another study evaluating more than 650 evaluable colorectal cancer samples found that neutrophil infiltration enhanced the prognostic significance of colorectal cancer infiltration by CD8⁺ T cells, which suggesting that they might improves survival in human colorectal cancer and be effective in promoting antitumor immunity ^[10].

Coronavirus disease 2019 morbid lung pathology shows that the lung autopsy of COVID-19 cases showed diffuse alveolar damage, and the cellular changes included massive infiltration of neutrophils, so the relationship between neutrophils and novel coronavirus pneumonia may be a potential research direction ^[11].

Conclusion

Neutrophils are an important component of the immune system and are necessary to protect the host from microbial infection. All processes in which neutrophils play a role, such as migration, recruitment, and clearance, are relevant to a variety of diseases, and their role in COVID-19 in particular worthy of further exploration.

Where to Get Human PB neutrophils?

AcceGen provides high quality Human PB neutrophils isolated from peripheral blood of fully consented IRB approved donors. Cells are cryopreserved immediately after isolation to ensure highest viability.

It is our pleasure to help relative researches to move forward. All the products of AcceGen are strictly comply with international standards. For more detailed information, please visit our product portfolio or contact inquiry@accegen.com.

Reference

[1] van Rees D J, Szilagyi K, Kuijpers T W, Matlung H L, van den Berg T K. Immunoreceptors on neutrophils. Semin Immunol, 2016, 28(2): 94-108

[2] Nauseef W M, Borregaard N. Neutrophils at work. Nat Immunol, 2014, 15(7): 602-611

[3] Kuhns D B, Priel D, Chu J, Zarember K A. Isolation and Functional Analysis of Human Neutrophils. Curr Protoc Immunol, 2015, 111: 7-23

[4] Gough P, Ganesan S, Datta S K. IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and  Function. J Immunol, 2017, 198(11): 4373-4382

[5] Lawrence S M, Corriden R, Nizet V. How Neutrophils Meet Their End. Trends Immunol, 2020, 41(6): 531-544

[6] Kajiume T, Kobayashi M. Human granulocytes undergo cell death via autophagy. Cell Death Discov, 2018, 4: 111

[7] Chen F ,  Min Y U ,  Zhong Y , et al. The role of neutrophils in asthma[J]. Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences, 2021, 50(1):123-130.

[8] Volder J D ,  Vereecke L ,  Joos G , et al. Targeting neutrophils in asthma: A therapeutic opportunity?[J]. Biochemical Pharmacology, 2020, 182:114292.

[9] Mas A ,  Vptb C . Neutrophils as an emerging therapeutic target and tool for cancer therapy[J]. Life sciences, 285:119952.

[10]Valeria, Governa, Emanuele, et al. The Interplay Between Neutrophils and CD8+T Cells Improves Survival in Human Colorectal Cancer[J]. Clinical Cancer Research, 2017.

[11] Zulfu A, Hamid S T ,  Elseed K A , et al. Coronavirus disease 2019 morbid pulmonary pathology: What did we learn from autopsy examinations? 2021.