James L. Mobley, Ph.D.

Neutrophils have been recognized for their role in the engulfment and destruction of invasive pathogenic microorganisms for more than a century. The critical role played by neutrophils in immune defense is underscored by the severity of diseases resulting from neutrophil absence or dysfunction, including Chronic Granulomatous Disease (CGD) and Leukocyte Adhesion Deficiency (LAD).¹ There are several features that make neutrophils the "special forces" of the innate immune response²:

1. They can be rapidly produced in very large numbers in response to a threat.
2. They are rapidly mobilized to the site of pathogen invasion.
3. They are capable of accurately identifying the enemy.
4. They carry multiple weapons capable of destroying the threat.

Production, Recruitment and Mobilization

In humans, 50-70% of white blood cells (leukocytes) in the bloodstream are neutrophils. These cells are mass-produced, fully differentiated, and rapidly replaced. They are generated in the bone marrow at a rate of 100 billion per day, and released into the circulation where they function or die within 1-2 days. They do not reproduce. In rodents, neutrophils constitute only 7-10% of circulating leukocytes, making the isolation and study of rodent peripheral blood neutrophils particularly challenging. The nature and consequence of this species-specific difference in neutrophil frequency is poorly understood.³

In all mammals, neutrophils are rapidly mobilized and recruited to the site of infection. The process of neutrophil recruitment has been thoroughly elucidated over the past twenty years and consists of three distinct steps. First, selectin-mediated rolling adhesion to the vascular endothelial cells slows the progression of the neutrophil in the circulation. Next, integrin-mediated firm adhesion promotes the arrest of forward motion. Finally, transendothelial migration allows the neutrophil to leave the circulation and crawl into the underlying infected tissue. Various chemoattractants, including leukotriene B₄ (LTB₄), act as homing signals to guide the extravasated neutrophils directly to the inflammatory site.⁴

Recognition

Once the neutrophil finds its way to the site of infection, its encounter with the pathogen is mediated by one or more pattern recognition receptors (PRRs) that bind to specific pathogen-associated molecular patterns (PAMPs) that are common to multiple invasive microorganisms.⁵ PRRs, in general, function in three different ways: Interacting directly with the PAMP on the cells surface, interacting directly with the PAMP in the cytoplasm, or interacting indirectly with the PAMP on the cell surface. The directly-binding cell surface PRRs recognize and respond to multiple ligands including bacterial lipopolysaccharide (via TLR4), peptidoglycan (TLR2), lipoteichoic acids (TLR2), bacterial DNA (TLR9), flagellin (TLR5), viral RNA (TLR3, TLR7, and TLR8), fungal B-glucan (Dectin-1) and N-formylated bacterial peptides (fMLP receptor). Intracellular PRRs include receptors that recognize bacterial peptidoglycan (NOD1) and muramyl dipeptide (NOD2). Other pattern recognition receptors are secreted from the cell (neutrophils or other immune cells) to encounter the pathogen at a distance. These include the pentraxin family members C-reactive protein and serum amyloid P, components of the complement cascade, and immunoglobulins. Once bound to their pathogen targets, these PRRs bind to the neutrophils through multiple distinct immunoglobulin Fc receptors or complement receptors. The interaction of PRRs with PAMPs promotes the phagocytosis of the pathogen, and the initiation of signal transduction cascades that trigger the activation of the arsenal of neutrophil anti-microbial weapons.

Defensive weaponry

Neutrophils, along with eosinophils, basophils, and mast cells, are collectively called "granulocytes" because of the high number of granules contained within their cytoplasm.⁶ Neutrophil primary granules, also called azurophilic granules, are thought to serve a lysosome-like function for the killing and digestion of pathogens contained within phagocytic vacuoles. Primary granules contain a plethora of anti-microbial agents including myeloperoxidase, phospholipase A₂, acid hydrolase, neutrophil elastase, lysozyme, cathepsin G, proteinase 3, iNOS, and several cationic peptides. These primary granules are rarely secreted, as their contents could be toxic to surrounding tissues. Neutrophil secondary granules, also called specific granules, contain alkaline phosphatase, NADPH oxidase, collagenase, lactoferrin, and cathelcidin (LL-37). Secondary granules are secretory in function, expelling their contents to the extracellular space. However, secondary granules also fuse with phagocytic vacuoles and primary granules, adding their contents to those of the primary granules. The NADPH oxidase contained within the secondary granules is required for the "respiratory burst," the generation of reactive oxygen species (ROS) including superoxide and hydrogen peroxide. Myeloperoxidase from the primary granules transforms hydrogen peroxide and chloride into hypochlorous acid, the active ingredient of chlorine bleach. Nitric oxide produced by iNOS reacts with superoxide to form microbicidal peroxynitrite. Once the infection has been eliminated, neutrophils undergo apoptosis, the process of programmed cell death that prevents the release of dangerous anti-microbial agents, thereby reducing damage to surrounding tissue. Apoptotic neutrophils are efficiently phagocytosed by macrophages that follow neutrophils into the inflammatory site.

Neutrophil extracellular traps (NETs)

One important component of the neutrophil antimicrobial response remained undiscovered until 2004 when Arturo Zychlinsky's lab described the production of NETs.⁷ As the name implies, NETs resemble web-like structures that the neutrophil weaves from DNA, histones, and antimicrobial macromolecules and extrudes into the extracellular space. The induction of NET formation, their composition, function, and removal are the subject of intense research.

Induction of NETs

The initial reports of NET formation in vitro used powerful activation stimuli (PMA, A23187, LPS) to promote NET formation over a 3-4 hour timeframe from highly purified human peripheral blood neutrophils. NET formation was initially assessed using fluorescent DNA-staining dyes for microscopy or by detection using microplate fluorescence readers.⁸ Later, DNA-independent assays were developed to measure other NET components that were released from NETs through the actions of nuclease.⁹ More recently, intravital microscopy has been employed to follow NET formation in living animals as it occurs in real time and under physiological conditions. Seminal studies from Paul Kubes et al. have shown that in mice, NET formation is dependent upon an interaction with platelets.¹⁰ High doses of LPS injected in vivo activate platelets to adhere to neutrophils and initiate NET formation. This process appears to be dependent upon platelet thromboxane A₂ (TXA₂), and can be inhibited by TXA₂ receptor antagonists.¹¹ This in vivo NET formation occurs more quickly (20 minutes) than PMA-induced NET formation in vitro, and does not result in the death of the neutrophil. Rather, the neutrophil body, devoid of a nucleus, is still able to move about in search of additional bacteria to engulf.

NET formation and composition

In the years since their discovery, the major structural components of NETs have been identified, including dsDNA, histones, myeloperoxidase (MPO), neutrophil elastase (NE), lactotransferrin, and defensin peptides.¹² Thus, it appears that NETs form as a result of the mixture of nuclear chromatin with the contents of the primary (alpha) granules. The exact mechanism of NET formation remains unclear, but several intermediate steps have been identified. Early in the process there appears to be a requirement of reactive oxygen species generated from the actions of NADPH oxidase. Neutrophils deficient in NADPH oxidase do not form NETs.¹³ Next, neutrophil elastase from the primary granules migrates to the nucleus where it degrades the linker histones (histone H1).¹⁴ MPO also migrates to the nucleus where it enhances the process of chromatin decondensation. Finally, the nuclear enzyme peptidylarginine deiminase 4 (PAD4) is engaged.¹⁵ PAD4 deiminates the chromatin core histones (H2A, H2B, H3, and H4), reducing their binding affinity for DNA, and thus promoting the unwinding of DNA from the core histones. At this stage the nascent NET is peppered with the antimicrobial macromolecules and extruded from the cytoplasm into the extracellular space.

NET function and clearance

The function of NETs was initially reported to be in the capture and killing of pathogenic microorganisms. Indeed, multiple bacterial species have been reported to die when co-cultured with NETs in vitro.¹⁶ It is likely that the NET serves as a matrix for the concentration of antimicrobial agents in a confined, localized area that maximizes lethality for pathogens while minimizing damage to the surrounding host tissues. Some bacterial species are resistant to the direct killing activity of NETs; viable bacteria are released when the NETs are treated with nuclease.¹⁷ However, the trapping of the bacteria in the NETs leads to enhanced phagocytosis by inflammatory macrophages, facilitating the indirect killing of these pathogens.

NETs contain multiple elements that if left in place too long, could induce damage to surrounding tissues or could be the targets of autoantibody formation. NETs are normally digested through the actions of serum DNAse 1 followed by the removal of NET particles by scavenger receptor-mediated phagocytosis by macrophages. Inappropriate or delayed clearance of NETs or NET particles has been associated with autoantibody formation in multiple autoimmune diseases including systemic lupus erythematosus (SLE).¹⁸ Human patients with SLE produce autoantibodies to dsDNA and histones, forming immune complexes that lead to kidney destruction. Mouse models of lupus also spontaneously produce autoantibodies against dsDNA and histones, and eventually die of kidney failure.¹⁹ At Cayman Chemical, we hypothesized that the lupus-prone mouse strain NZBWF1 might also make autoantibodies against other NET components, and that these antibodies might be useful tools for detecting NETs by ELISA or immunohistochemistry. Indeed, we were able to produce monoclonal antibodies from NZBWF1 mice that recognize dsDNA, histones H2A, H2B, H3, and H4, myeloperoxidase, lysozyme, cathepsin G, and mCRAMP, the mouse version of LL-37 (see page 6). Many of these spontaneously generated antibodies are directed against conserved elements of their target antigens, and therefore are capable of recognizing NET components from multiple species.

PAD4

In the formation of neutrophil extracellular traps, the nuclear enzyme peptidylarginine deiminase (PAD4) modifies histones in such a way that it allows the dissociation of tightly packaged DNA from the core histone octamer. This is a critical step in NET formation. Mice deficient in PAD4 expression cannot form NETs and are susceptible to bacterial infection. Thus, the primary role of PAD4 in neutrophils appears to be in host defense, a function not recognized until 2010.²⁰ Prior to this report, most immunologists were already familiar with PAD4, not for its beneficial role in killing bacteria, but rather for its contribution to the pathophysiology of rheumatoid arthritis.

PAD4 catalyzes the post-translational modification of arginine to citrulline within peptides or proteins.²¹ By removal of the terminal guanidine group, arginine loses a positive charge, resulting in the disruption of ionic interactions with negatively charged macromolecules, including DNA. This is thought to be the mechanism whereby PAD4 promotes chromatin decondensation. This process must be tightly regulated; unrestrained PAD4 activation leading to widespread arginine deimination could also disrupt intramolecular interactions within individual proteins, promoting protein unfolding and loss of function, and increasing susceptibility to proteolytic degradation. Furthermore, by transforming positively charged arginine to a charge neutral citrulline, PAD4 alters the ability of processed peptides containing citrulline to bind to the major histocompatibility (MHC) Class II proteins on antigen presenting cells.²² This effect could result in the misidentification of "self" peptides as "non-self" by T lymphocytes, leading to a T cell-dependent autoimmune response directed towards the citrullinated protein.

In 1994, Schellekens et al. first reported that patients with rheumatoid arthritis produce autoantibodies against citrulline-containing proteins.²³ This observation led to the development of highly selective assays for anti-citrullinated protein/peptide antibodies (ACAP) that are diagnostic for human rheumatoid arthritis. It also led to the identification of several proteins to which these autoantibodies are directed and include citrullinated fibrinogen, vimentin, filaggrin, and fibrin.²⁴ In addition, a subset of RA patients produce autoantobodies directed against the PAD4 enzyme itself.²⁵

Cayman Chemical makes a variety of research tools for the study of PAD4 activity, including recombinant human PAD4, a PAD4 inhibitor screening kit, two small molecule PAD4 inhibitors Cl-amidine and F-amidine (see page 9-10). Cayman also sells PAD4-citrullinated human fibrinogen, and will soon begin to produce citrullinated vimentin and histone H3 along with a fluorescent chemical probe that can be used to assess the presence of citrulline within target proteins (see page 10). Finally, Cayman produces the only commercially available assay kit for detecting anti-PAD4 autoantibodies from human plasma or serum (see page 9). As new mouse models of rheumatoid arthritis are developed that recapitulate the human disease role for PAD4, citrullinated peptides, and anti-citrullinated protein antibodies, Cayman Chemical will be there to develop the tools necessary to make research possible in this exciting new area of biology.

Figure 1. Stimulated neutrophil with NETs and some trapped Shigella bacteria (orange). Colored scanning electron micrograph. © Max Planck Institute for Infection Biology

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