At least two populations of tissue macrophages exist: tissue-resident cells that are embryonically derived and a monocyte-derived population. Their functions may be discrete.
Macrophages are plastic and will change in response to environmental cues through pattern-recognition receptors and other sensors. These changes are regulated primarily at the epigenetic and transcriptional levels.
The M1/M2 paradigm does not fully characterize macrophage heterogeneity.
Synovial macrophages produce a variety of pro-inflammatory cytokines that contribute to synovitis and can be targeted with therapeutic agents.
Transcriptional profiling of synovial macrophages from patients with rheumatoid arthritis might provide insight into disease activity and response to therapy.
Introduction
Metchnikoff described macrophages in the early 1900s, but synovial macrophages were not identified until nearly 60 years later ( Fig. 10.1 ). Three populations of synoviocytes were described based on electron microscopy and were termed type A, type B, and type C in the early 1960s. It is now known that type A cells were synovial macrophages, type B were synovial fibroblasts, and type C were an undetermined population. Further studies in the 1980s and early 1990s refined the classification of synovial macrophages with the use of immunohistochemistry and known antibodies to antigen-presenting cells. These studies were the first to suggest that the synovial macrophage population may be heterogeneous based on cell surface expression of proteins; location (lining vs. sublining); and production of cytokines, chemokines, and matrix metalloproteinases. Additionally, the origin of synovial macrophages was linked to monocytes derived from the bone marrow, based on studies using radiation chimeras of beige mice. Taken together, these fundamental studies provided the basis that the synovial macrophages were derived from hematopoietic progenitors and represented a heterogeneous population in both normal and inflamed synovium. The goal of this chapter is to provide an up-to-date review of macrophage biology and a revision of the current dogma of the mononuclear phagocyte system (MPS) as it relates to the pathogenesis of rheumatoid arthritis (RA).
Steady-State Development of Synovial Macrophages
Early studies in the 1960s and 1970s shaped our understanding of macrophage biology. , These studies determined that adult monocytes develop as precursors in the bone marrow and then enter the circulation to replenish macrophages in tissue through the use of radiolabeled monocyte progenitors. Nevertheless, more recent studies challenged these assumptions through the use of radiation chimeras, parabiotic mice, and lineage-tracing experiments. The current paradigm is that the vast majority of macrophages are “tissue-resident,” developing during embryogenesis and self-renewing in most tissues in the absence of inflammatory stimuli or severe depletion. ,
Initial studies suggested that, in adults, a common monocyte/dendritic cell (MDP) progenitor in the bone marrow leads to the development of monocytes, macrophages, and dendritic cells. Recent studies, however, suggest that MDPs may also develop into other types of hematopoietic cells, such as lymphocytes. , The administration of nonlethal irradiation to induce death of hematopoietic cells and their precursors followed by administration of donor bone marrow (radiation chimeras) revealed populations of macrophages, including synovial macrophages, Langerhans cells, and microglia, that were resistant to irradiation and remained host origin, whereas the monocyte population was derived from donor hematopoietic cells. Moreover, parabiotic mice that share the same circulation showed that only a subset of macrophages such as heart, gut, and dermis exhibited a mixed population, whereas Langerhans cells, microglia, and alveolar macrophages were exclusively derived from the parent mouse. These studies were the first to provide support for an alternative hypothesis to the MPS regarding monocyte replacement and macrophage turnover in adult mice.
Studies using radiation chimeras and parabiotic mice demonstrated that monocytes only populate tissue-resident macrophages in a few organs during steady-state conditions in adult mice. The origin of the adult tissue-resident macrophage, however, was unknown until the generation of fate-mapping and lineage-tracing mice. These mice express tamoxifen-induced Cre recombinase (Mer-cre-Mer) that enzymatically removes a stop codon flanked by lox sequences (floxed) on a reporter gene such as green fluorescent protein or yellow fluorescent protein, thus resulting in traceable fluorescence on the cells of interest. Runt-related transcription factor 1 (Runx1) is required for the development of erythro-myeloid precursors (EMP) and hematopoietic stem cells (HSC) from the hemogenic endothelium. The YS-derived precursors are restricted to days E7.0 to 7.5, while expression at E8.5 is limited to the definitive hematopoiesis stage, which involves fetal monocyte differentiation into macrophages. Runx1-Mer-cre-Mer mice, which have a tamoxifen-inducible cre recombinase flanked by two mutated estrogen receptors and a floxed neo into the Runx1 gene downstream of the proximal (P2) promoter were crossed with fate mapping (reporter) mice and treated with tamoxifen to induce Cre recombination at day E7.0 versus E8.0. , These mice revealed that microglia were the predominant cell that fluorescently labeled at E7.5 and persisted throughout adulthood. In contrast, any macrophages that were positive at E7.5 lost their label during embryogenesis, which suggests that they were being replaced through nonlabeled precursors. , , Consistent with this idea, the numbers of fluorescent positive monocytes and macrophages by the reporter gene increased progressively, whereas the numbers of positive microglia were negligible. , ,
Another study took advantage of embryos that lacked c-myb, a gene required for definitive hematopoiesis. These mice were deficient for F4/80 low CD11b hi myeloid cells but still retained the F4/80 bright CD11b low macrophages, which were originally described as resident macrophages. , These data suggested that fetal macrophages arise from the c-myb-independent pathway in the yolk-sac (YS), whereas the majority of hematopoietic precursors require c-myb expressing progenitors. In support of this idea, these investigators used Tie2-Mer-cre-Mer mice, because Tie2 is highly expressed in hemogenic endothelial progeny such as EMPs and fetal HSCs. , The addition of tamoxifen at E7.5, E8.5, or E9.0 revealed a differential labeling pattern of embryonic monocytes and macrophages in tamoxifen-treated Tie2-Mer-cre-Mer mice. , With induction at E7.5, adult tissue-resident macrophages were more labeled when compared with nonmacrophage leukocytes; at E8.5, the proportion of labeled tissue-resident macrophages and leukocytes were comparable; and at E9.5, the leukocytes were more labeled than the tissue-resident macrophages in the tamoxifen-treated Tie2-Mer-cre-Mer mice. , Taken together, these results suggest that the tissue-resident macrophage precursors are formed early in embryogenesis (i.e., E7.5) and resemble the EMPs. Moreover, the late EMPs or fetal HSCs require c-myb for the development of tissue-resident macrophages with the exception of microglia and some Langerhans cells. Further studies using c-kit-Mer-Cre-Mer mice supported the idea that embryonic HSC is crucial for the development of tissue-resident macrophages. Nevertheless, it is possible that the loss of c-myb induces a redundant pathway that functions independent of c-myb.
More recent studies indicated that there are two decoupled waves of EMPs at days E7.5 (primitive hematopoiesis) and E8.5 (the transient definitive stage); the former is responsible for the development of the microglia, whereas the latter travels through the circulation and supports the development of fetal monocytes that traffic to the liver and then to the whole embryo through the circulation. , , , In this work, YS macrophages were depleted using an anti–colony stimulating factor (CSF) antibody injected at E6.5. The fetal monocyte population was not affected, and the tissue-resident macrophage populations including the microglia were able to recover. These data suggest that YS macrophages are not required for tissue-resident macrophage development and that there is another CSF-independent pathway. This concept is supported by the fact that YS macrophages were replaced in the embryo via fetal monocytes with the exception of microglial cells and a minor fraction of Langerhans cells over time, as observed through the injection of tamoxifen at early E8.5 or late E14.5 time points in Csf-Mer-cre-Mer and the Runx-Mer-cre-Mer mice or through the use of S100A4-cre mice, which labels myeloid cells after YS development when crossed with a reporter mouse. , , , Moreover, the fetal monocytes may be independent of HSC development because Flt3-cre/reporter mice, which label HSCs in adult mice, failed to label fetal HSCs sufficiently. Later stage EMPs, however, require c-myb to form the FL monocytes. , , , ,
Taken together, these data suggest that primitive hematopoiesis starts at E7 in YS and gives rise to EMPs that directly differentiate into microglia and a portion of Langerhans cells, bypassing the monocyte stage. Spatially and temporally regulated waves of hematopoiesis, including a transient definitive wave, also produce EMPs, which arise from YS at day E8 to E8.5; differentiate into fetal monocytes and progenitors for myeloid cells; and migrate via embryonic blood circulation to other tissues, including a fetal liver, to start the definitive hematopoietic stage. During this stage, almost all embryonic tissue macrophages are developed before the production of HSCs ( Fig. 10.2 ).
It was well established in the late 1980s that human synovial macrophages are heterogeneous, but the origin of these cells was unknown. Similar to other tissue macrophage populations in mice, murine synovial macrophages exist as monocyte-derived and YS-derived (tissue-resident) in a steady state ( Fig. 10.3 ). The monocyte-derived synovial macrophages are a minor population, have a high turnover rate, require M-CSF, are sensitive to irradiation, express class II major histocompatibility complex (MHC), and are poor phagocytes. In contrast, the YS-derived, tissue-resident macrophages self-populate, do not require M-CSF, are insensitive to irradiation, do not express class II MHC, and are phagocytic. These studies document the origin of murine synovial macrophages, but it is still unknown whether human synovial macrophages develop in a similar manner to mice.
Transcriptional Regulation of Synovial Macrophages
Macrophage function is defined by the expression of genes that are influenced by ontogeny, stimuli, and environment ( Fig. 10.4 ). Macrophage genes are regulated by a complex network of transcription factors; proteins that recognize specific DNA sequences or motifs; and the cis-regulatory elements, such as enhancers, that they bind. Multiple transcription factors have been proposed to specify macrophage fate based on the prevalence of their binding sites in macrophage-specific enhancers. Foremost among them is PU.1, which binds a large proportion of enhancers in macrophages and will be further discussed later in this section. PU.1 binds enhancers in both peritoneal macrophages and splenic B cells, but only macrophages demonstrate co-binding with C/EBP and AP-1 factors. C/EBPα and C/EBPβ have been implicated in macrophage development from HSCs. C/EBPα is critical early on in hematopoiesis for commitment to the myeloid lineage, whereas C/EBPβ is expressed only upon differentiation from the macrophage progenitors.
When compared with other myeloid cells, including monocytes and neutrophils, macrophages demonstrate increased binding of transcription factors in the MAF family. Similarly, MAFB is important for microglia maturity: MAFB-null microglia fail to adapt the transcriptional profile associated with the adult brain. MAF family transcription factors (TFs) may be necessary for terminal macrophage differentiation in tissues by regulating self-renewal. In addition, the interferon regulatory factor (IRF) family motif, particularly as a composite with PU.1 (PU.1-IRF), is often found in macrophage enhancers. , , The macrophage-specific factors described above do not bind in isolation; these TFs are often bound in combination with each other.
PU.1, a member of the ETS family, represents a special class of TFs known as pioneers . , Pioneers are master regulators that bind thousands of sites across the genome and are capable of increasing the chromatin accessibility of a region. In this way, they establish enhancers at which other TFs can settle. PU.1 binding occurs in multiple hematopoietic cell types but is best known for its role in myeloid lineage specification. The PU.1 motif is found in all macrophage progenitors from monocytes back through to HSCs. PU.1 is necessary for the deposition and maintenance of macrophage enhancers and binds in combination with other macrophage-specific TFs. Mutations between mouse strains that lead to the disruption of PU.1 motifs affect PU.1 binding, cofactor binding (such as CCAAT enhancer binding protein [C/EBP]α), and enhancer accessibility. , In normal hematopoiesis, the expression of PU.1 rather than GATA1 leads to commitment to the myeloid lineage over the erythroid lineage. , By upregulating myeloid genes and remodeling the enhancer landscape, PU.1, in combination with ectopic expression of C/EBPα or C/EBPβ, can force transdifferentiation of fibroblasts, B cells, and T cells into macrophage-like cells. Not only are lineage-specifying TFs like PU.1 important for macrophage development, but they also determine response to stimuli.
Macrophages are adapted to turn on specific genes in response to stimuli. When bone marrow–derived macrophages (BMDMs) are treated with lipopolysaccharide (LPS), which is commonly used for macrophage stimulation, the majority of activated enhancers are previously primed. , Only a small subset of regions, known as latent enhancers , are opened de novo with stimulation. Macrophages derived from human monocytes also demonstrate limited changes to the enhancer landscape when stimulated with LPS. Similar results have been seen with other stimuli, such as Kdo2-lipid A (KLA) and TNF, where the stimulated enhancer landscape was largely composed of pre-existing enhancers. , , Different sets of enhancers, however, are associated with different stimuli. Primed enhancers are bound by lineage-specific TFs, such as PU.1 and C/EBPα. , , , With stimulation, stimulus-dependent TFs, such as NF-κβ and STAT, are recruited to a subset of these enhancers. , , , In this way, a macrophage-specific, stimulus-specific response is elicited. To investigate genome-wide binding in these experiments, high-throughput sequencing assays must be performed on macrophage populations. For this reason, much of these studies have been performed on macrophages cultured in vitro to obtain sufficient numbers and controlled conditions. Nevertheless, these macrophages may not fully reflect the function of macrophages in vivo in the local environment of the tissue. Although the underlying principles still apply, the in vitro response cannot capture the heterogeneity across macrophage populations.
Macrophages are often described as a plastic cell type because they are capable of adapting different functions depending on environmental signals. Since macrophages reside in almost every tissue of the body, they are exposed to a wide variety of local environments. For example, macrophages in the peritoneum respond to retinoic acid in the environment, which leads to the induction of GATA6 for their characteristic gene expression. GATA6 is a TF exclusively expressed in peritoneal macrophages compared with other tissue-resident macrophages, and the GATA motif is enriched at peritoneal-specific macrophage enhancers. , When macrophages are extracted from the peritoneum and grown in culture, they lose their distinctive enhancer landscape, but it is partially rescued by treatment with retinoic acid. Similarly, microglia exhibit the highest expression of Mef2c, and microglia-specific enhancers are enriched for MEF2 motif. , Transforming growth factor (TGF)-β may be one of the key signals in the microglia environment, leading to the development of mature microglia and the establishment of the adult enhancer landscape. , , Other tissue-specific TFs implicated by the macrophage enhancer landscape include peroxisome proliferator-activated receptor γ (PPARγ) in the lung; liver X receptor α (LXRα) in liver and spleen; retinoid X receptor (RXR) in lung and spleen; and runt-related transcription factors (RUNX) in intestinal macrophages. These TFs bind in combination with general macrophage factors, such as PU.1, to regulate tissue-resident macrophage genes. A shared environmental signal across two macrophage populations, such as exposure to erythrocyte turnover in liver and spleen, may lead to a shared TF, but the collective binding of TFs in response to the assortment of signals in the unique environment leads to a distinct enhancer landscape regulating tissue-specific expression.
No study has compared the transcriptional landscape of synovial macrophages with other tissue-resident macrophage populations. One might predict, however, that synovial macrophage gene expression will be determined by a combination of cell-type-specific factors with tissue-specific factors that may overlap with those already seen or not, depending on the signals in the joint environment. Many of the studies listed in previous sections supporting macrophage-specific TFs were performed on mouse cells, but homologous proteins are likely to play a similar role in humans. Because it is difficult to obtain macrophage samples from human tissues, many of these factors have yet to be validated in humans.
Synovial Macrophage Production of Cytokines and Chemokines
Macrophages are plastic, which is not only governed by their origin (i.e., embryonic vs. monocyte-derived) but also by their microenvironment, especially during disease initiation, pathogenesis, and resolution. , The vast majority of our knowledge regarding synovial macrophages is attributed to studies that examined human synovial macrophages via dual immunohistochemistry or in situ hybridization of tissue sections from RA synovial tissue, synovial fluid macrophages in culture, and/or conversion of RA peripheral blood monocytes to macrophages in culture. , , For most studies, synovial tissue was retrieved via joint replacement surgery of patients. Thus, these studies were unable to examine the synovium in early RA or in patients experiencing flares, although some groups were able to use percutaneous synovial biopsies or arthroscopic biopsies. Moreover, synovial tissue from osteoarthritis patients was commonly used as a comparison, which may not be ideal because osteoarthritis has an inflammatory component. Nonetheless, the co-expression patterns of the synovial lining and sublining macrophages with transcription factors and cell signaling molecules such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), janus kinase/signal transducers and activators of transcription (JAK/STAT), C/EBP, c-Jun N-terminal kinase (JNK), extra-cellular regulating kinase (ERK), and p38, anti-apoptopic or proapoptotic proteins or cytokines/chemokines helped show pivotal and topographical roles for synovial macrophages during RA pathology. , ,
In culture, macrophages may be unstimulated or activated with pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), such as conventional and joint-specific agonists to the toll-like receptor pathways, the exogenous addition of TNF or IL-1β, and/or culture with macrophage (M)-CSF or granulocyte-macrophage (GM)-CSF on plastic dishes. , , Through the studies, synovial macrophages were established to be one of the central producers of the cytokines TNF, IL-1, IL-6, IL-8, IL-10, IL-12, IL-18, IL-15, GM-CSF, and M-CSF, as well as chemokines such as CC motif ligand (CCL)3, CCL5, chemokine (CXC motif) ligand (CXCL)1, CXCL8, CCL2, and IL-8 in the joint. , , Although collectively the cell culture experiments helped identify cell-specific production of individual cytokines and chemokines found in the synovium, they are fraught with potential issues that have only been recently identified due to technologic advances (i.e., bulk RNA sequencing and single-cell RNA sequencing). It has now become clear that macrophages express a different transcriptional profile in culture from those in tissue, due to the complex nature of the in vivo microenvironment. Synovial macrophages are continually in contact with other synovial cells and a milieu of cytokines and chemokines that could not be replicated in culture. , Moreover, culture conditions could never capture the heterogeneity of synovial macrophages, but would instead favor one or more of the individual population(s). The future of understanding synovial macrophages ex vivo will be through the development of organoid cultures and/or 3D micromass cultures that contain both synovial fibroblasts and macrophages under physiologic conditions, which develop a synovial lining-like structure.
The M1 and M2 Paradigm Revisited
Over the past 30 years, macrophage biologists have created a paradigm that mimics the classical T helper (Th)1/Th2 mechanism for T-cells but can be applied to macrophages. Terms such as activation and polarization are commonly used to describe a particular state or phenotype of macrophage. One study described two phenotypes of macrophages: one activated by INFγ and called classically activated (M1), and one stimulated with IL-4 and referred to as alternatively activated (M2). Cultured macrophages were classified into M1 and M2 phenotypes using mice that were biased towards Th1/Th2 readouts such as C57BL/6 and BALBc, respectively. Typically, M1 (classically activated) macrophages treated with INFγ, LPS, GM-CSF, and TNF produce TNF, IL-1, IL-16, IL-23, IL-12, type I INF, inducible nitric oxide synthase (iNOS), and CXCL9, 10, and 11 and express class II MHC, CD80, CD86, CCR1, and CCR5 and promote Th1 responses.
In contrast, M2 (alternatively activated) macrophages are induced by IL-4, IL-13, M-CSF, immune complexes, IL-10, and glucocorticoids, resulting in the expression of IL-4, IL-10, CCL16, 17, 18, 22 and 24, Chi3l3, arginase, Ym-1 and Relmα, CD163, CD206, and CCR3. To work with the limited flexibility of the M1/M2 paradigm, others established additional nomenclatures such as M2b (regulatory macrophages) and M2c (wound healing macrophages). For example, immune complexes and Toll-like receptor (TLR) activation, apoptotic bodies, cyclic adenosine monophosphate (cAMP), prostaglandin E2, TGF-β, or IL-10 induce a remission/regulatory macrophage phenotype.
There are many drawbacks to the M1/M2 nomenclature. The basis for this nomenclature was initially attributed to the generation of M-CSF or L929 treated bone marrow–derived macrophages or peritoneal cells in culture for 7 days, which does not parallel any in vivo macrophage population. In vivo, the local environment is flushed with stimulants affecting numerous transcriptional and translational programs that contain aspects of both classical and alternatively activated cells. Seminal studies by investigators showed that by adding one stimulant at a time, the transcriptional readouts were not able to fit into one single category. Taken together, the M1/M2 macrophage paradigm is strictly a well-defined molecular event that is highly reproducible under controlled in vitro conditions but has little or no relevance in vivo. , , Thus, in vivo macrophages may display characteristics or transcriptional profiles of pro-inflammatory as well as profibrotic and/or proresolution signatures , and more likely exist within a spectrum of activation states.
Macrophages in Murine Models of Inflammatory Arthritis
The hypertrophy of the synovial lining, as well as the increased cellularity of the synovial sublining, may be attributed to increased recruitment of immune cells (efflux of monocytes), reduced egress, local proliferation, or lack of death. Animal models of RA-like disease that recapitulate various aspects of disease activity, which occur in RA patients (for a comprehensive review, please see reference 80), are effective to understand the mechanism behind the hyperplasia of the synovium (see Chapter 32 ). There are two main spontaneous models of arthritis (K/BxN and TNF-transgenic [Tg] mice). Previous studies have shown that mice expressing the KRN T-cell receptor (TCR) in the context of the class II MHC Allele H-2k (Ag7) develop a severe, spontaneous, symmetric, and erosive arthritis that resembles human RA. K/BxAg7 (C57Bl/6 background) or K/BxN mice also develop noninfective endocarditis, another feature similar to humans with RA. It was then determined that the Ag7 class II MHC allele presented endogenous glucose-6-phosphate isomerase (G6PI) peptides that were recognized as pathogenic by the KRN TCR. Importantly, 64% of patients with RA produce anti-G6PI antibodies, suggesting a similarity in the pathogenesis between the K/BxAg7 model and human disease. The TNF transgenic model was generated through overexpression of the human TNF gene lacking post-transcriptional regulatory elements, allowing for continued expression of TNF. The development and course of arthritis are dependent on the numbers of copies of the transgene but do not require lymphocytes as TNFTg RAG −/− mice still develop arthritis.
There are also inducible models of inflammatory arthritis. Collagen-induced arthritis (CIA) is a chronic model of arthritis induced by immunization with collagen in complete Freund’s adjuvant. This elicits a loss of tolerance to native collagen resulting in bone destruction and recruitment of immune cells. Both K/BxN and CIA models recapitulate human disease with a chronic and destructive pathology involving the innate and adaptive immune system. , Further, K/BxN and CIA can be used as passive models through transfer of sera-containing anti-G6PI antibodies or anti-collagen antibodies in the K/BxN serum transfer model (STIA) or collagen antibody induced model (CAIA), respectively. Both result in passive, resolving inflammatory arthritis in the recipient mice. The disease in STIA mice represents the effector phase of RA, is independent of lymphocytes, and consists of an initiation, a developmental/propagation, and a resolution stage. Innate and adaptive immune components including B cells, T cells, neutrophils, mast cells, macrophages, complement factors, inflammatory cytokines (IL-1, TNF, IL-17), and Fc receptors contribute to the development of spontaneous arthritis and/or experimentally induced arthritis models.
Monocyte and Macrophage Contribution to Synovial Hyperplasia
Monocytes are divided into at least two main populations: classical and nonclassical monocytes. Classical monocytes have a shorter life span and convert into nonclassical monocytes in bone marrow or the circulation. In mice, all monocytes are CD45 + CD11b + CD115 + , but classical monocytes are further characterized as Ly6C + CD62L + CCR2 + CD43 − , whereas nonclassical monocytes are Ly6C − CX3CR1 + CD62L − CCR2 − CD43 + . In humans, the classical monocyte population is considered CD45 + CD11b + CD14 ++ CD16 − HLADR + CCR2 + , while the nonclassical monocytes are CD45 + CD11b + CD14 + CD16 + HLADR + CX3CR1 + . There are also intermediate populations that are in a state of conversion from a classical to nonclassical monocyte. Although the exact roles for each population remain under investigation, it is clear that both populations of monocytes are involved in response to an insult. Nevertheless, the nonclassical monocytes may have an additional role as they are thought to patrol the vasculature to help maintain endothelial cells integrity and potentially limit the factors and cells which extravasate into tissues through the endothelium.
Clodronate-loaded liposomes are commonly used to eliminate monocytes and tissue macrophages. When delivered systemically, clodronate-loaded liposomes deplete all bone marrow monocytes, circulating monocytes, splenic monocytes and macrophages, liver macrophages, and some kidney macrophages. The synovium is spared, however, due to its low vascularity. Systemic treatment of clodronate-loaded liposomes prevented STIA and CIA because local delivery to the knee suppressed AIA and IL-1/mBSA induced arthritis. Similarly, mice with a mutation in M-CSF (thus, deficient in monocytes and some tissue macrophages) or who lack GM-CSF fail to develop CIA and IL-1/mBSA induced arthritis. ,
Treatment with an antagonistic M-CSF or GM-CSF antibody or an oral inhibitor of M-CSF also prevents CIA through the recruitment of monocytes. , Mice lacking CCR2, which have reduced numbers of classical monocytes, are still as equally susceptible to STIA as wild-type mice. , Further, antibody-mediated depletion of CCR2 does not affect the development of STIA, and a CCR2-deficient model of TNFTg arthritis (CCR2 −/− TNFTg) displays an exacerbated form of arthritis. Replacement of nonclassical monocytes restores the development of STIA following monocyte depletion with clodronate-loaded liposomes, whereas transfer of classical monocytes does not, and reduction in nonclassical monocytes via CX3CR1 depletion also leads to less STIA. , These findings suggest that nonclassical monocytes, not classical monocytes, are the critical population for development of inflammatory arthritis. Nevertheless, successful treatment of TNFTg arthritis with anti-TNF antibodies is associated with a reduced efflux of classical monocytes and enhanced classical monocyte apoptosis in the synovium of mice without impacting egress into the lymph node, indicating that although classical monocytes may not be critical to disease initiation, they contribute significantly to pathology and progression of disease. Although monocytes may be essential for disease development, macrophages are critical for the remission phase as the deletion of both populations of synovial macrophages dramatically delays the spontaneous remission of inflammatory arthritis in mice. Taken together, these data demonstrate that individual cell populations have unique roles throughout the course of disease in inflammatory arthritis, which is influenced by the specific microenvironment created by the arthritis model.
Recently tissue-resident macrophages proliferated and maintained their niche. The increased numbers of macrophages were examined in bone marrow chimeric mice and following EDU treatment. There was no change in the rate or numbers of proliferating monocytes or macrophages in the STIA model. Moreover, mice lacking p21, a cell cycle inhibitor, led to increased STIA through hyperactivation of synovial macrophages. Although these data do not rule out a contribution of monocyte and macrophage proliferation to arthritis development in other models of RA-like disease, the vast majority of studies indicate that the enhanced cellularity of the synovium is attributed to increased infiltration of leukocytes.
A lack of apoptosis has also been suggested to contribute to the development and sustainment of inflammatory arthritis. The apoptotic machinery has long been associated with determining and executing the fate of a cell. Cells undergo apoptosis via two central but distinct pathways, an “extrinsic” pathway that requires binding of death ligands (Fas Ligand) to their cognate receptors (Fas) on the cell surface, and an “intrinsic” pathway in which mitochondria play a critical role ( Fig. 10.5 ). The extrinsic pathway is suppressed by Flip, which binds to caspase 8, sequesters it, and prevents caspase 8 autocatalysis. The intrinsic pathway is regulated by the Bcl-2 protein family, which is divided into antiapoptotic (Bcl-2, Bcl-xL, Mcl-1) and proapoptotic (Bax, Bak, Bim) members. While there are over 10 Bcl-2 homology (BH3)-only proteins (such as Bad, Bid, Bmf, Noxa, or Puma) or multi-BH domains (Bak and Bak), which are considered the inducers or executioners of mitochondrial apoptosis respectively, none of them result in spontaneous autoimmune-specific phenotype following deletion except Bim. Increased expression of Flip, Mcl-1, and Bcl-2 and reduced expression of Bim have been reported in the human synovium.
