Re-Examining Neutrophil Participation in GN

Abstract

Significant advances in understanding the pathogenesis of GN have occurred in recent decades. Among those advances is the finding that both innate and adaptive immune cells contribute to the development of GN. Neutrophils were recognized as key contributors in early animal models of GN, at a time when the prevailing view considered neutrophils to function as nonspecific effector cells that die quickly after performing antimicrobial functions. However, advances over the past two decades have shown that neutrophil functions are more complex and sophisticated. Specifically, research has revealed that neutrophil survival is regulated by the inflammatory milieu and that neutrophils demonstrate plasticity, mediate microbial killing through previously unrecognized mechanisms, demonstrate transcriptional activity leading to the release of cytokines and chemokines, interact with and regulate cells of the innate and adaptive immune systems, and contribute to the resolution of inflammation. Therefore, neutrophil participation in glomerular diseases deserves re-evaluation. In this review, we describe advances in understanding classic neutrophil functions, review the expanded roles of neutrophils in innate and adaptive immune responses, and summarize current knowledge of neutrophil contributions to GN.

Despite advances in understanding the pathogenesis of various forms of GN, therapy has not advanced past nonspecific inhibition of immune and inflammatory responses. Those therapies induce complete or partial remissions in 50% or fewer patients, and they are associated with significant side effects. A more complete understanding of the effectors of glomerular injury will identify targets for new therapies. Early studies using animal models of immune complex and anti–glomerular basement membrane (GBM) GN recognized neutrophils as a necessary cellular component of glomerular injury.¹ At the time those studies were performed, the prevailing view was that neutrophils were short-lived, terminally differentiated cells serving as nonspecific effectors; were present briefly during the acute phase of inflammation; and possessed little or no synthetic capability. Consequently, the majority of the nephrology research community shifted focus to the contribution of B cells, monocytes/macrophages, T cells, dendritic cells, and intrinsic glomerular cells.^2,3 The past two decades have witnessed the discovery of exciting new information about neutrophils, indicating that survival is regulated by the external milieu, expanded mechanisms for microbial killing exist, a number of cytokines and chemokines are expressed and released, cells of both innate and adaptive immune systems interact with and are activated by neutrophils, and lipids that promote resolution of inflammation are produced.^4–6 In light of those expanded capabilities, we believe the participation of neutrophils in the development of various forms of GN deserves re-evaluation. In this review, we summarize new findings related to classic neutrophil functions, describe expanded functional activities in innate and adaptive immune responses, and review current knowledge of neutrophil participation in GN. Those expanded functional capabilities suggest that neutrophils may be more important contributors to various forms of GN, and identifying those contributions may provide new therapeutic targets.

New Understanding of Classic Neutrophil Functions and Their Role in GN

Figure 1 illustrates the series of coordinated events leading to neutrophil elimination of bacteria at sites of infection, including adhesion to activated vascular endothelial cells, transmigration across the vascular wall, chemotaxis to the site of infection, phagocytosis of bacteria, and generation and release of chemicals that kill those organisms.⁷ To accomplish those tasks, neutrophils express >100 cell surface receptors to sense their microenvironment and regulate their cellular responses (Table 1). This section summarizes recent advances in understanding those coordinated events relevant to neutrophil participation in glomerular inflammation.

Figure 1.

The sequential steps of classic neutrophil recruitment from the vasculature to tissue in response to infection are shown. Sentinel cells are stimulated by PAMPs or DAMPs to release chemokines, cytokines, and lipid mediators that activate vascular endothelial cells to express selectins and chemokines. Neutrophils undergo capture and fast rolling through the interaction of selectins on endothelial cells with their ligands on neutrophils. Selectins and chemokines induce integrin-mediated slow rolling and neutrophil arrest. Neutrophils undergo integrin-mediated crawling to a site for transmigration. Transmigration at endothelial cell junctions is mediated by a number of additional adhesion molecules. Release of proteolytic enzymes allows neutrophils to enter the extravascular space. Extravascular neutrophils follow a chemoattractant gradient to the site of infection. Pathogens are attacked by phagocytosis and by release of NETs. Microbes within phagosomes are killed by a combination of granule contents and generation of ROS. Infiltrating neutrophils are eliminated by apoptosis.

Table 1.

Neutrophil cell surface receptors

Neutrophil Cell Surface Receptors
G-protein coupled receptors
Chemoattractant receptors: FPR1 (FPR), FPR2 (FPRL1), FPR3 (FPRL2), BLT1 (LTB4R), BLT2 (LTB4R), PAFR, C5aR
Chemokine receptors: CXCR1 (human), CXCR2, CXCR4, CCR1, CCR2, CX3CR1
Fc receptors
FcγRs: FcγRI, FcγRIIA (human), FcγRIIB (inhibitory), FcγRIII (mouse), FcγRIIIB (human), FcγRIV (mouse)
Fcα-receptors: FcαRI (human)
Fcε-receptors: FcεRI, FcεRII
Adhesion receptors
Selectins and selectin ligands: L-selectin, PSGL-1, ESL-1, CD44
Integrins: LFA-1 (αLβ2), Mac-1 (αMβ2), VLA-4 (α4β1)
Cytokine receptors
Type I cytokine receptors: IL-4R, IL-6R, IL-12R, IL-15R, G-CSFR, GM-CSFR
Type II cytokine receptors: IFNAR, IFNGR, IL-10R
IL-1R family: IL-1RI, IL1RII, IL-18R
TNFR family: TNFR1 (p55), TNFR2 (p75), Fas, LTβR, RANK, TRAIL-R2, TRAIL-R3
Innate immune receptors
TLRs: TLR1, TLR2, TLR4, TLR5, TLR6, TLR7 (?), TLR8, TLR9
C-type lectins: Dectin-1, Mincle, MDL-1, Mcl, CLEC-2
NOD-like receptors: NOD2, NLRP3
RIG-like receptors: RIG-I, MDA5
Adenosine receptors
A1, A2A, A2B, A3
ITIM-bearing inhibitory receptors
CD300a, CD300f, CD200R, CEACAM1, CLEC12a, CLEC4a, LILRB2, LILRB3, PILRa, SIGLEC-5, SIGLEC-9, SIRL-1, SIRP-a

Neutrophils express a large number of cell surface receptors including G-protein coupled receptors, Fc receptors, adhesion receptors, cytokine receptors, innate immune receptors, adenosine receptors, and ITIM-bearing inhibitory receptors.^151–153

Neutrophil Recruitment

Recruitment of circulating neutrophils is a multistep cascade triggered by the release of pathogen-associated molecular pattern molecules (PAMPs) during infection or damage-associated molecular pattern molecules (DAMPs) during tissue injury (Figure 1). PAMPs and DAMPs interact with pattern recognition receptors on the surface (Toll-like receptors [TLRs] and C-type lectin receptors) and in the cytoplasm (Nod-like receptors, RIG-I–like receptors, and TLR-7, -9, and -10) of sentinel cells, including macrophages and dendritic cells, causing them to release chemokines, cytokines (e.g., IL-1β and TNFα), and lipid mediators that initiate neutrophil recruitment.^8,9 Those mediators induce vascular endothelial cells to increase surface expression of selectins and enhance access to selectins through changes in the thickness and composition of the glucosaminoglycan endothelial surface layer.^10–12

Contact between neutrophils and activated endothelial cells in postcapillary venules leads to neutrophil capture and fast rolling through the interaction of E- and P-selectins on endothelial cells with P-selectin glycoprotein ligand–1 (PSGL-1), E-selectin ligand–1 (ESL-1), and CD44 on neutrophils.^11,13,14 Rolling exposes neutrophils to chemokines immobilized on the endothelial cell surface.¹² The combination of intracellular signals generated by selectins and chemokine receptors leads to a conformation change of the integrin, LFA-1 (CD11a/CD18), that results in an intermediate affinity for ICAM-1.¹⁴ The combination of interaction of LFA-1 with ICAM-1 and selectins with PSGL-1 results in slow rolling. Continued exposure to chemokine- and selectin-mediated signaling induces a high affinity conformation of LFA-1, resulting in neutrophil arrest. Additionally, those signals stimulate increased neutrophil surface expression of the integrin Mac-1 (CD11b/CD18) through granule exocytosis.^15,16 After arrest, neutrophils undergo intraluminal crawling toward a site favorable for transmigration through the interaction of Mac-1 with ICAM-1.^12,17 Transmigration through the endothelial cell layer occurs primarily at endothelial cell junctions and is mediated by a number of adhesion molecules, including MAC-1; platelet/endothelial cell adhesion molecule–1; junctional adhesion molecules –A, –B, and–C; and endothelial cell selective adhesion molecules, CD99 and CD99L2.^12,14 Once through the endothelial cell layer, neutrophils use proteolytic enzymes, such as gelatinase, released from intracellular granules to traverse the vascular basement membrane and enter the extravascular space.¹⁸ Extravascular neutrophils are exposed to a sequential cascade of chemoattractants that stimulate chemotaxis and chemokinesis to the location of infection or inflammation.¹⁹ Long-distance recruitment is usually mediated by “intermediate target” chemoattractants, leukotriene B₄ and IL-8, that abruptly switch to “end-target” chemoattractants, formylated peptides and C5a, at 100–150 µm from the source of inflammation.^19,20 Neutrophils arrive at the site of inflammation in two waves. The first wave is initiated by recruitment, described above. In the second wave, commonly referred to as swarming, long-distance neutrophil recruitment is driven by LTB₄ produced by neutrophils recruited in the first wave.²¹ This is one of many examples of amplification loops initiated by neutrophils.²²

This description of neutrophil recruitment was derived from studies of the systemic vasculature and recruitment was typically induced by exposure to soluble inflammatory mediators. Alternative methods of neutrophil recruitment are used in the specialized vascular beds of glomerular capillaries (Figure 2).^23–26 P-selectin on platelets adherent to endothelial cells can serve as a bridge between endothelial cells and neutrophils, a process termed “secondary capture.”¹¹ Capture of circulating neutrophils by platelets has been described in murine anti-GBM GN.^23,27 Immune complex deposition can lead to neutrophil capture through Fcγ receptor (FcγR) recognition of IgG whereas Mac-1 integrins maintain adhesion under flow conditions.²⁴ The requirement of FcγR and MAC-1 expression for glomerular neutrophil recruitment and development of proteinuria was described in a murine model of anti-GBM GN.^24,28,29 Passive transfer of sera from patients with SLE to CD11b/CD18-deficient mice expressing human FcγRIIA and FcγRIIIB on neutrophils showed that FcγRIIA mediates neutrophil accumulation and glomerular injury.³⁰ The role of endothelial cell selectins in glomerular neutrophil recruitment remains unclear. Evidence for and against selectin-mediated neutrophil recruitment in anti-GBM GN has been published, and an inhibitory role has even been described.^{23,25,27,31,32}

Figure 2.

Three alternative methods of glomerular neutrophil recruitment are shown. “Secondary capture” involves platelets serving as a bridge between endothelial cells and neutrophils. A second method of neutrophil capture occurs by direct interaction with immune complexes through FcγRs or complement receptors. A third method involves neutrophil retention by slowing their crawling along glomerular capillaries. Increased neutrophil retention requires direct interaction between monocytes and neutrophils, production of TNFα by patrolling monocytes, and integrin expression by neutrophils.

Multiphoton microscopy of hydronephrotic mouse kidneys showed that neutrophils and monocytes constantly patrol normal glomeruli by crawling along capillaries (Figure 2).²⁶ Induction of anti-GBM GN increased neutrophil and monocyte retention within the glomerulus by slowing their travel along the capillary bed, but did not induce transmigration. Prolonged glomerular dwell time of both neutrophils and monocytes was also induced by administration of antibodies against myeloperoxidase (MPO) and by activated glomerular T cells, but not by noninflammatory podocyte injury.²⁶ Increased neutrophil retention required direct interaction between monocytes and neutrophils, as well as production of TNFα by patrolling monocytes.³³ Prolonged neutrophil transit time through inflamed glomeruli required Mac-1 expression, whereas prolonged monocyte transit time was dependent on LFA-1, Mac-1, and CX₃CR1.^26,33

The effects of these alternative methods of glomerular neutrophil recruitment on neutrophil responses, such as enhanced respiratory burst activity and granule exocytosis, transcriptional activation, and the rate of apoptosis, have not been determined. The discovery that neutrophils undergoing prolonged glomerular transit time demonstrated enhanced reactive oxygen species (ROS) generation and CXCL1 release, but did not produce TNFα, suggests that differences in some of those responses will be found.³³

Complement Activation

Activation of the C cascade by immune complexes has long been identified as a mechanism for neutrophil recruitment and glomerular cell injury.^28,34–37 An additional role of C activation in ANCA-associated vasculitis (AAV) has been described. C depletion or C5a receptor blockage was reported to protect anti-MPO–treated mice from developing necrotizing crescentic GN.^38,39 ANCA-stimulated neutrophils were found to activate the alternative C pathway, leading to generation of C5a.⁴⁰ C5a recruited additional neutrophils and primed those neutrophils for an enhanced respiratory burst and degranulation in response to ANCA.^41,42 Patients with active AAV have high levels of C5a, and other C factors.⁴³ A clinical trial to evaluate the safety and efficacy of an oral inhibitor of the C5a receptor (CCX168) in AAV was completed in 2016 (NCT01363388), but study results have yet to be posted (clinicaltrials.gov).

Mechanisms of Microbial Killing

ROS and antimicrobial granule components cooperate to kill invading microbes engulfed by neutrophils into phagosomes (Figure 1).⁴⁴ Extracellular release of those microbial toxins leads to tissue injury. The molecular events leading to ROS generation by the neutrophil NADPH oxidase have been extensively studied.⁴ Signals generated by microbial invasion or tissue injury, including TLR agonists, chemokines, proinflammatory cytokines, and proinflammatory lipids enhance neutrophil production of ROS in response to activation signals, a process termed “priming.”^45–48 In addition to enhanced ROS generation, primed neutrophils demonstrate enhanced chemotaxis, exocytosis, adhesion, and transcriptional activity in addition to delayed apoptosis. Neutrophil priming is postulated to play a critical role in development of GN in AAV.^39,40,49 Priming by TNFα also enhances neutrophil recruitment by immune complexes.^47,50

Evidence for induction of, and protection against, glomerular injury by ROS has been reported. Suzuki et al. reported that neutrophil interaction with glomerular immune complexes through FcγRs led to ROS-dependent NF-κB activation, TNFα overexpression, and further neutrophil recruitment.⁵¹ Feith et al. reported that albuminuria after 72 hours of the heterologous phase of anti-GBM nephritis was dependent on neutrophil ROS generation.⁵² Hypohalous acid, but not H₂O₂, was shown to increase albumin permeability of isolated rat glomeruli, and intrarenal generation of hypohalous acid by renal artery injection of MPO and H₂O₂, but not H₂O₂ alone, induced proteinuria in rats.^53,54 Odobasic et al. reported that MPO contributes to ROS-mediated glomerular injury in the heterologous phase of anti-GBM GN, but attenuates injury during the autologous phase by suppressing T cell proliferation and cytokine production.⁵⁵ Mice genetically deficient in neutrophil NADPH oxidase failed to develop proteinuria after administration of anti-GBM antibodies.²⁶ On the other hand, Schreiber et al. reported that mice genetically deficient in NADPH oxidase components (gp91^phox, p47^phox) demonstrated accelerated ANCA-mediated crescentic GN, due to diminished ROS-mediated downregulation of IL-1β.⁵⁶

The role of neutrophil granule components in glomerular disease is most completely understood in AAV-mediated GN. Target antigens for ANCA (MPO, PR3, and possibly LAMP-2) are stored in neutrophil granules and secretory vesicles.^57–59 Target antigens in granules are not accessible to circulating ANCA; however, neutrophil priming increases their plasma membrane expression, allowing ANCA to stimulate neutrophil degranulation and ROS production through neutrophil FcγRs.^60–63 Depletion of circulating neutrophils also protects mice from anti-MPO–induced GN.⁶⁴ The extent of neutrophil ROS production and degranulation depends on the level of ANCA expression on plasma membranes.⁶⁵ Using a mouse model of AAV, Schreiber et al. showed that glomerular cytokine generation, neutrophil and monocyte infiltration into glomeruli, and crescent formation were dependent on neutrophil release of granule proteases.⁶⁶ Lu et al. reported that in vitro neutrophil-mediated endothelial cell injury by ANCA was induced by serine proteases, not ROS.⁴²

Neutrophil serine proteases (neutrophil elastase, cathepsin G, and proteinase 3) have been studied in other models of GN. Feith et al. showed that neutrophil release of serine proteases was required for albuminuria at 24 hours in a model of anti-GBM nephritis.⁵² Schrijver et al. used C57BL/6J,bg/bg (beige) mice, in which neutrophils are genetically deficient in neutrophil elastase and cathepsin G but demonstrate normal ROS generation, to examine their role in anti-GBM GN.⁶⁷ Beige mice failed to develop proteinuria despite equivalent glomerular neutrophil accumulation and normal neutrophil respiratory burst activity.⁶⁷ Suzuki et al. reported that administration of an inhibitor of neutrophil elastase prevented development of proteinuria, hematuria, and crescent formation in a rat model of anti-GBM nephritis.⁶⁸ Both neutrophil elastase and cathepsin G degrade GBM in vitro.⁶⁹ Neutrophil elastase has also been detected in glomeruli and urine from patients with crescentic GN, MPGN, and membranous nephropathy.^70,71 Another neutrophil granule component, gelatinase (MMP9), was shown by immunohistochemistry and zymography to be present in glomeruli of patients with several types of GN, including AAV, IgA nephropathy, acute postinfectious GN, and lupus nephritis (LN).⁷² Infusion of elastase and other MMPs into isolated rat glomeruli increased albumin permeability.⁷³

The studies summarized above suggest that generation of ROS and release of granule enzymes participate in neutrophil-mediated glomerular injury, although their relative contribution depends on factors yet to be identified. The understanding of the mechanisms of injury and the granule components responsible is incomplete. Proteomic analysis of the four subsets of neutrophil granules (secretory vesicles; gelatinase or tertiary granules; specific or secondary granules; and azurophilic or primary granules) identified >800 protein constituents.^57,58,74 A number of those newly discovered granule proteins have been shown to contribute to induction and resolution of the inflammatory response.^75–79 The large number of neutrophil granule constituents identified suggests that a focus on granule proteases is too narrow.

Expanded Neutrophil Functions and Potential Role in GN

Neutrophil Heterogeneity and Plasticity

The life span of neutrophils in the circulation was thought to be limited to a few hours by constitutive apoptosis. Pillay et al. challenged that concept and suggested the normal life span may be up to several days.⁸⁰ This observed increase in life span, however, was subsequently challenged on methodologic grounds.⁸¹ Neutrophil apoptosis does not occur at a fixed rate, but can be increased or decreased at sites of inflammation by various cytokines, growth factors, and bacterial products.⁸² Thus, the neutrophil contribution to severity and resolution of inflammatory responses is influenced by the rate at which they die. It is unclear if regulation of neutrophil apoptosis during glomerular inflammation resembles that found in other inflammatory conditions. Less than 20% of neutrophils recruited to the glomerulus in a model of immune complex GN were reported to undergo apoptosis.⁸³

Subpopulations of neutrophils with different surface molecules and densities demonstrate unique functional characteristics.^84,85 For example, a low-density subpopulation of neutrophils that express programmed death receptor 1 ligand (PDL-1) shows immunosuppressive properties in HIV-infected patients.^86,87 Immunosuppressive neutrophils also have been identified in patients with autoimmune diseases, cancer, and pregnancy.^88–93 Neutrophil plasticity is also demonstrated by their ability to acquire properties of dendritic cells or macrophages, including antigen presentation, upon exposure to specific cytokine combinations.⁸⁴ Thus, subpopulations of resting and stimulated neutrophils may perform important immune regulatory functions in GN, although a role of neutrophil subpopulations in pathophysiology remains to be determined.

Neutrophil Extracellular Traps

In 2004, a new antimicrobial mechanism was described in which neutrophils form an extracellular network of chromatin fibers containing globular domains of antimicrobial proteins derived from intracellular granules, termed neutrophil extracellular traps, or NETs.⁹⁴ NET formation is stimulated by activation of Fc receptors, TLRs, and receptors for C components, IL-8, TNFα, IFNs, and by exposure to gram-positive and gram-negative bacteria and fungi.^94–97 NET formation occurs by a unique form of cell death, termed NETosis, in which nuclear and granule membranes dissolve, the nuclear content decondenses into the cytoplasm, and the plasma membrane ruptures releasing strands of chromatin decorated with granular proteins.⁹⁸ Bacteria, fungi, and parasites bind to NETs through a charge interaction, leading to their exposure to high concentrations of antimicrobial proteins, including histones, defensins, lysozyme, and serine proteases.⁹⁹ Although NETs possess antibacterial activity in vitro, their ability to kill bacteria in vivo has proven difficult to demonstrate.¹⁰⁰

A number of reports suggest that NETs are involved in autoimmune and glomerular diseases, including AAV and LN.¹⁰¹ Kessenbrock et al. showed that neutrophils from normal subjects primed by TNFα in vitro demonstrated robust NET formation upon exposure to ANCA containing IgG fractions and mouse monoclonal anti-PR3.¹⁰² NETs were shown to be present in glomeruli in renal biopsy samples from patients with rapidly deteriorating renal function due to AAV.^102,103 Recently, multiphoton microscopy identified NETs in about 20% of glomeruli in murine anti-GBM GN.¹⁰⁴ However, those NETs were present only transiently, and their disruption with DNase did not alter the development of proteinuria.¹⁰⁴

Neutrophils from patients with SLE are also more likely to form NETs which contain antigens (dsDNA, histones) recognized by lupus autoantibodies, particularly when they are stimulated with immune complexes.^105–108 In addition to providing antigens recognized by autoantibodies, components of NETs stimulate dendritic cells to release IFN-α through TLR-9 and enhance T cell activation by antigen through an unknown mechanism.^106–109 Hakkim et al. showed that a subset of patients with SLE had impaired degradation of NETs, and those patients were significantly more likely to have LN.¹¹⁰ Leffler et al. showed that patients with SLE with impaired degradation of NETs had significantly lower serum levels of C3 and C4, and those NETs were able to directly activate either the classic or lectin C pathways.¹¹¹

Microparticles

Another new mechanism by which neutrophils may control bacterial invasion is through release of microparticles.¹¹² Microparticles are a member of a group of vesicles (that includes exosomes, ectosomes, microvesicles, and shedding microvesicles) released from all activated or dying cells that contribute to cell-cell communication. Timar and colleagues reported that incubation of isolated human neutrophils with opsonized bacteria stimulated the rapid release of microparticles able to inhibit growth of both opsonized and nonopsonized bacteria through bacterial aggregation.¹¹² Neutrophil-derived microparticles with the same surface markers were present with bacterial aggregates in the blood of patients with bacteremia. Those microparticles showed higher expression of granule antibacterial proteins. Neutrophil-derived microparticles have also been reported to induce pro- and anti-inflammatory macrophage phenotypes, and they participate in venous thrombogenesis.^113–115

Evidence for participation of neutrophil-derived microparticles in glomerular diseases is sparse, but suggestive. Microparticles derived from neutrophils and platelets are present in the plasma in patients with active vasculitis.¹¹⁶ Anti-neutrophil cytoplasmic antibodies were reported to stimulate the release of microparticles from neutrophils.¹¹⁷ Those microparticles attached to endothelial cells in an integrin-dependent manner, where they stimulated increased adhesion molecule expression, ROS generation, cytokine release, and thrombin generation.

A number of studies described an increased concentration of microparticles derived from endothelial cells, platelets, and leukocytes in patients with SLE and antiphospholipid syndrome.^118–120 Nielsen et al. reported that the concentration of cell-derived microparticles was decreased in SLE, although the ability of those microparticles to bind annexin V differed from normal individuals.¹²¹ Because some microparticles contain nucleic acids, the ability of microparticles to contribute to immune complex formation has been examined.¹²² Nielsen et al. showed that circulating microparticles from patients with SLE contained increased IgG, IgM, and C1q, compared with patients with rheumatoid arthritis and systemic sclerosis or healthy controls.¹²³ Ullal et al. reported that anti-DNA and anti-nucleosomal antibodies bound to microparticles derived from cultured monocytes, T cells, and neutrophilic cells.¹²⁴ Thus, cell-derived microparticles may participate in the generation of autoantibodies and in the formation and size of immune complexes that induce LN.

Neutrophils Interact with Innate and Adaptive Immune Cells

Neutrophils are now known to contribute to both innate and adaptive immune responses through direct and indirect interactions with monocytes, endothelial cells, T- and B-lymphocytes, natural killer (NK) cells, and dendritic cells. Neutrophils influence the immune response through synthesis and secretion of cytokines and chemokines (Table 2) and release of granule contents.^75,125,126 Serine proteases, azurocidin, α-defensins, and LL-37 are granule components that induce monocyte adhesion and chemotaxis and activate endothelial cells to increase expression of adhesion molecules.^127–129 Those proteins have also been reported to induce release of monocyte-attracting chemokines by endothelial cells and tissue macrophages, including MCP-1, MIP-1, and IL-8.^130,131 Serine proteases proteolytically modify cytokines and chemokines and their receptors, resulting in modified activity.⁷⁹ Azurocidin and PR3 disrupt endothelial cell integrity leading to increased vascular permeability and edema.^128,132,133 LL-37 has both proinflammatory and anti-inflammatory activities through regulation of TLRs on neutrophils and monocytes.¹³⁴

Table 2.

Cytokines and chemokines produced by neutrophils that contribute to experimental and human GN

Cytokine/Chemokine	Animal Model of GN	Human GN
CC chemokines
CCL2 (MCP1)126	LN154	AAV,155 Crescentic GN,156 LN157
CCL3(MIP1α)126	LN158	Crescentic GN,156 SLE159
CCL4(MIP1β)126		Crescentic GN156
CCL18126,160		AAV161
CCL20126	Anti-GBM,162 IgAN163
CXC chemokines
CXCL1126	AAV164	IgAN,165 LN166
CXCL5126	Anti-GBM167	AAV167
CXCL8 (IL-8)126	Immune Complex GN168	AAV164,169
CXCL9126	Anti-GBM162
CXCL10126		LN157
CXCL 12126	LN154
CXCL 13126	LN170	AAV,171 LN170,172
Colony-stimulating factors
G-CSF125	AAV173	AAV174
GM-CSF125	AAV173
Proinflammatory cytokines
IL-1α125,175		AAV,176 IgAN,177 MPGN,178 LN178
IL-1β125,175,179	AAV,66,180 LN181	AAV,182,183 IgAN,177 LN,178,184 MPGN178
IL-6125,185,186	Anti-GBM,187 LN188–190	AAV,183 IgAN,191 LN,178 MPGN178
IL-17125,192,193	AAV,194 Anti-GBM,195,196 LN195	LN197
IL-18125,198,199	LN200	AAV,201 LN,197,202,203
MIF125	LN204	Crescentic GN,205 FSGS,205 LN,205 MPGN205
Anti-inflammatory cytokines
IL-1ra125,206	Anti-GBM207	IgAN,177 LN208
IL-10a125,209–212		LN/SLE202,213
IL-4125,214	Anti-GBM215	MN216
TGFβ1125,217	Anti-GBM218	IgAN219
Immunoregulatory cytokines
IL-12125,209,220,221	LN222,223	LN/SLE197,202,224
IL-21125	LN225
IL-27125,226	Anti-GBM227
IFN-α125,228,229	LN230,231	FSGS,232LN/SLE229,233–236
IFN-β125		FSGS,232,237 MPGN238
IFN-γ125,229,239	LN222,240–242	AAV,216 FSGS,232 MPGN,216 LN/SLE229,243
TNF superfamily members
TNF-α244,245	Anti-GBM180	AAV,176,182 IgAN,191 LN/SLE,178,246 MPGN178
APRIL247,248		LN/SLE249
BAFF247,250	LN251,252	LN/SLE249,253
FasL126	Anti-GBM254	LN255
TRAIL126		LN256

Neutrophils are capable of expressing and producing a number of cytokines, although in smaller quantities than other immune cells. Several cytokines/chemokines have been implicated in experimental and human GN. However, the contribution from neutrophils is not well described and requires further inquiry. IgAN, IgA nephropathy; MPGN, membranoproliferative GN; MN, membranous nephropathy.

^aEvidence for and against production by neutrophils.

Several antimicrobial peptides released from neutrophil granules play an active role in recruiting and activating dendritic cells. Those peptides, termed ‘alarmins’, include LL-37, α-defensins, and lactoferrin.^76,135–137 There is now evidence that neutrophils interact in a network with dendritic cells, T-cells, and NK cells to modulate the immune response in both infection and chronic inflammation.^84,138,139 That modulation includes recruitment of T helper 1 and T helper 17 cells to infected sites by neutrophil-derived chemokines.¹⁴⁰ In concert with dendritic cells, neutrophils potentiate the release of GM-CSF and IFN-γ by NK cells, which, in turn, promotes neutrophil survival and priming of ROS production.^139,141,142 Upon migration into tissues, neutrophils undergo transcriptional activation resulting in production of proinflammatory cytokines, a number of which have been shown to participate in GN (Table 2).^75,77,143 Although the ability of neutrophils to produce those cytokines and chemokines is a fraction of monocyte capacity, the large number of migrating neutrophils may provide physiologically relevant quantities. The contribution of glomerular neutrophils to the production of cytokines and other agents that regulate innate and adaptive immunity requires further investigation.

Neutrophils and Resolution of Inflammation

Emerging evidence suggests that neutrophils play a role in the resolution of inflammation.¹⁴⁴ In the late stages of an inflammatory response, neutrophils switch their eicosanoid biosynthesis from proinflammatory leukotriene B₄ to anti-inflammatory lipoxin A₄, which inhibits neutrophil recruitment.¹⁴⁵ Wu et al. demonstrated increased levels of lipoxin A₄ in the glomeruli and leukocytes of patients with poststreptococcal GN.¹⁴⁶ Poststreptococcal GN is usually a self-limited illness, and high levels of lipoxin A₄ may contribute to disease resolution. Lipoxin A₄ attenuates PDGF-mediated mesangial proliferation in cell culture and decreases neutrophil recruitment in an animal model of anti-GBM nephritis.^147,148 Neutrophils also contribute to the resolution of inflammation through the production of resolvins, which can inhibit neutrophil transendothelial migration and tissue infiltration.¹⁴⁵ The role of resolvins in GN has not been examined.

The expanded functional capabilities of neutrophils outlined in this review suggest that neutrophils may contribute to GN through initiation of disease, causing direct injury to glomerular cells or the filtration barrier, regulating activity of other cells of the innate and adaptive immune systems, or initiating resolution programs. Defining the role neutrophils play in development and resolution of various forms of GN will enhance our understanding of the systems biology of immune glomerular injury. The complex interactions between neutrophils and other immune cells and intrinsic glomerular cells suggests that the time for examining the role of individual cell types in GN may be coming to an end. Examining the complex cellular interactions within the glomerulus is likely to expand our understanding of the pathophysiology of GN and to identify new therapeutic targets. Examples of that potential were recently provided by two studies examining the effect of selective inhibition of neutrophil granule exocytosis in animal models of disease. Uriarte and colleagues inhibited neutrophil granule exocytosis in vivo by intravenous infusion of a cell-permeable peptide that blocked SNARE protein interactions.¹⁴⁹ Treatment with that peptide significantly reduced acute lung injury induced by immune complex deposition. Recently, high through-put screening identified small molecules that inhibited neutrophil exocytosis in vivo by interfering in the interaction between the small GTPase Rab27a and its effector JFC1.¹⁵⁰ Administration of one of those molecules to mice with endotoxin-induced systemic inflammation resulted in reduced neutrophil extravasation into tissues. Those examples illustrate the potential for new therapeutic avenues that will be identified by further understanding of the complex cellular interactions leading to glomerular inflammation.

Disclosures

D.J.C. receives research support from Mallinckrodt Pharmaceuticals.