Role of Leukotriene B₄ Receptors in Rheumatoid Arthritis

Abstract

The purpose of this review is to summarize the role that murine models of arthritis are playing in the understanding of human rheumatoid arthritis and how leukotriene B₄ (LTB₄) is emerging as an important target in this field. Both the collagen-induced arthritis (CIA) model and the K/BxN serum transfer arthritis model have contributed to outline the potential mechanisms involved in inflammatory arthritis. Indeed, the CIA model has contributed to the development of effective anti-TNF and anti-IL-1β based treatments for RA that are currently in the clinic. Many recent studies in mouse models have suggested a critical role for LTB₄ and its receptors in the development of inflammatory arthritis. Inhibitors of LTB₄ biosynthesis as well as LTB₄ receptors are protective in mouse models of RA and mice deficient in the LTB₄ biosynthetic enzymes or LTB₄ receptors are resistant to disease development suggesting several promising targets for RA in this pathway.

Take home message

• Mouse models of RA have provided a great deal of information on the mechanisms involved in human RA and led to the development of effective therapies and are likely to uncover additional therapeutic opportunities

• Chemoattractants and cytokines form important amplification loops for perpetual joint inflammation in RA

• Inhibition of inflammatory cell recruitment to the rheumatoid synovium represents a potential target for RA treatment.

• LTB₄ and its receptors play a critical role in the recruitment of leucocytes to the inflammatory sites. Mice lacking either the enzymes involved in LTB₄ biosynthesis or the LTB₄ receptors are completely protected from the development of RA.

• Unraveling the mechanisms of LTB₄/BLT1 and LTB₄/BLT2 axis and structure/function of BLT1 and BLT2 will provide important insights into identification of novel and dual antagonists for blocking their function.

INTRODUCTION

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that affects ~1% of the world population. Despite lack of consensus on what initiates RA in humans, many recent developments point to a role for the innate immune system in the pathogenesis of RA [1–3]. Many concepts developed in experimental animal models have begun yielding effective therapeutics for arthritis such as anti TNF-α and anti IL-1β based therapies that are currently leading the way[4, 5]. These therapies have been effective, but subgroups of RA patients do not respond to them. Another treatment regimen that showed great promise was the use of cyclooxygenase-2 (COX-2) inhibitors [6]. However, these had to be withdrawn due to increased risk of cardiovascular complications in patients taking the drug [7]. A clear understanding of the pathogenic mechanisms in mouse models will likely provide additional therapeutic targets for the treatment of RA. One of these potential leads involves leukotriene B₄ (LTB₄), a potent lipid inflammatory mediator and a strong chemoattractant for neutrophils. LTB₄ mediates it’s effects through two G-protein coupled receptors (GPCRs), BLT1 (high affinity) and BLT2 (low affinity) [8, 9]. Recent data from animal models and arthritis patients demonstrate a critical role for LTB₄ and its receptors in the progression of RA and suggests potential new targets for treatment. Here, we discuss briefly the available mouse models of RA and their use in the demonstration of an important role for LTB₄ and its receptors in the development of RA.

Mouse Models of Arthritis

Collagen-induced arthritis (CIA] has been the most widely used model of arthritis initiated by intradermal immunization with type II collagen [10]. The immunization protocol varies due to a difference in susceptibility of the strains of mice, which is thought to be due to MHC class linking. DBA/1 (H-2^q) and B10-RIII (H-2^r) mice develop disease following immunization with collagen in Complete Freund’s Adjuvant (CFA) for the first injection and using incomplete Freund’s adjuvant (IFA) for the booster two to three weeks later. CIA in mice has been shown to have several features in common with human RA. In particular, the cytokine requirement and the effects of cytokines on the development and progression of the disease appear similar to human RA [11]. In this regard, TNF-α and IL-1β appear critical in the development and progression of RA[12]. IL-2, IL-6, IL-12, and IL-18 act as positive modulators and IL-4 and IL-10 are negative modulators of the disease activity [13–15].

Two other mouse models of RA have also attracted significant attention in the recent years [16, 17]. The K/BxN model based on a T-cell receptor (KRN] transgenic mouse which produces a T-cell repertoire that recognizes and makes auto antibodies to the ubiquitous glycolytic enzyme, glucose-6-phosphate isomerase (GPI) and develops an aggressive form of arthritis [18]. In this case disease may be transferred with serum or purified antibodies. Disease development in this model is T and B-cell independent while neutrophils and mast cells are required for joint inflammation [2, 19]. An interesting recent observation in this model suggests that histamine and serotonin control vascular leakage and incite joint inflammation by deposition of immune complexes formed against systemic antigens [20]. Modification of the immunization protocol allowed CIA development in C57BL/6 mice [16]. In this case use of CFA in both primary and booster injections with collagen is a critical determinant for disease development. Each of these models has been used to show a critical requirement for LTB₄ in the development and progression of arthritis.

Leukotriene B₄ and its Receptors

Leukotriene B₄ is a potent mediator of inflammation derived from arachidonic acid by the sequential actions of 5-lipoxygenase (5-LO) and LTA₄ hydrolase (Fig. 1) [21]. LTB₄ acting through its receptors (BLT1) can cause chemotaxis, degranulation, adhesion and enhance the survival of neutrophils. Although BLT1 was long known to be a neutrophil chemoattractant receptor, recent studies identified BLT1 expression on macrophages [22], smooth muscle cells [23], endothelial cells [24], activated T-cells [25] and mast cells [26] considerably expanding the potential role of LTB₄. Recent experiments from our laboratory demonstrated functional expression of BLT1 on both mature and immature dendritic cells and having a direct effect in the control of adaptive immune responses [27]. Knockout mice for BLT1 generated in several laboratories have been instrumental in defining a critical role for this receptor in diverse inflammatory diseases such as atherosclerosis [22] asthma, autoimmune uveitis and arthritis (see below). In contrast, the function and biological activities of BLT2 are completely unknown. BLT2 has been shown to be expressed widely in humans, with the spleen and peripheral blood leukocytes showing the highest expression [9]. However, murine BLT2 expression has been difficult to determine with several laboratories reporting varying results [26, 28, 29]. Although a BLT1/BLT2 double deficient mouse line was recently reported, complete understanding of the unique functions of BLT2 will not likely emerge until it can be targeted.

Figure 1. The role of LTB₄ in the development of arthritis.

LTB₄, a pro-inflammatory lipid mediator, is synthesized from the arachidonic acid pathway involving the biosynthetic enzymes 5-lipoxygenase (5-LO), Five-Lipoxygenase Activating Protein (FLAP) and LTA₄ Hydrolase (LTA₄H) via 5HPETE and LTA₄ as intermediates. Arthritis might be initiated by infection mediated activation in the joints and/or from the formation of auto-antibodies leading to deposition of immune complexes (IC) in the synovium. The immune complexes activate mast cells and cause mast cell degranulation, which further leads to the influx of inflammatory cells through vasodilation and other mediators. Interplay of synovial cells and the incoming leukocytes leads to production of cytokines and activation of the complement cascade. Amplification loops between the cytokines and chemoattractants like LTB₄ and other chemokines sets up perpetual joint inflammation. All of the innate immune cells such as dendritic cells, macrophages, neutrophils and mast cells are capable of producing LTB₄ by the induction of complement or cytokines. Since all of these cells express LTB₄ receptors they set up further amplification loops in the inflamed joints. The solid lines represent response to and broken lines represent production of indicated mediators.

Role of Leukotriene B₄ Receptors in Arthritis

A role for LTB₄ in rheumatoid arthritis was suggested by several observations over the past two decades. Neutrophils from RA patients undergoing methotrexate therapy displayed both acute and chronic suppression of LTB₄ synthesis ex vivo [30].LTB₄ is a potent chemoattractant of neutrophils and promotes the adhesion of neutrophils to vascular endothelium, which promotes arthritis development [31]. In a recent study, a significant increase in the mRNA levels of BLT1 and BLT2 was seen in the joint tissues and cells from RA patients relative to OA patients suggesting a role for these receptors in RA [32].

Development of targeted gene deletions in mice and of antagonists to the enzymes responsible for the production of LTB₄ or to the receptors allowed for a better understanding of the requirement for LTB₄ in arthritis (Table 1). In early studies the LTB₄ receptor antagonists, CP-105,696 was shown to greatly reduce disease severity in an IL-1α accelerated CIA model [33]. Independent studies with two other LTB₄ receptor antagonists also yielded similar results in IL-1α accelerated CIA model [34]. In an LPS accelerated CIA model, use of a FLAP inhibitor SA6541 reduced the severity of disease [35]. Studies in mice deficient in the LTB₄ pathway quickly followed the antagonist experiments. FLAP knock-out mice were put through the IL-1α accelerated CIA model and were found to have a 73% reduction in disease severity, which was associated with a 23% decrease in disease incidence. FLAP heterozygous mice were shown to have a 37% decrease in disease severity and a similar decrease in disease incidence as the FLAP knock-out mice, suggesting that FLAP is a potential drug target [36]. Though this pathway looked like a good target for arthritis treatment, attempts to use the antagonists in clinical trials of arthritis apparently failed.

Table 1.

Role of LTB₄ and its receptors in murine arthritis models

Mouse Strain	Model of Arthritis	Experimental Results	References
DBA/1LacJ	IL-1 accelerated CIA	Complete protection against disease development by administration of BLT1 antagonist CP-105,696	(35)
DBA/1	IL-1 Accelerated CIA	A 38% reduction in disease severity by administration of BLT1 antagonist LY293111Na or CGS25019C	(36)
DBA/1	LPS-accelerated CIA	Reduced severity of disease by administration of FLAP inhibitor SA6541	(37)
DBA/1	IL-18 accelerated CIA	5-LO inhibitor MK-886 reduced the severity of disease	(41)
FLAP−/− DBA/1	IL-1 Accelerated CIA	Reduced incidence and severity of disease in FLAP−/− mice relative to WT mice	(38)
C57BL/6	K/BxN	Complete protection by preventive administration and reduced severity by therapeutic treatment with 5-LO inhibitor L-739,010 and with BLT1 antagonist CP-105,696	(39)
BLT1−/− and BLT1/BLT2−/− C57BL/6	CIA	Complete protection from disease development in BLT1−/− and BLT1/BLT2−/− mice. Similar levels of auto antibody (anti-C II) production in receptor deficient animals	(31)
5-LO−/− and LTA4H−/− C57BL/6	K/BxN	Complete protection from disease development in 5-LO−/− and LTA4H−/− mice. Adoptive transfer of WT neutrophils is sufficient for disease development in 5LO−/− mice	(20)
BLT1−/− C57BL/6	K/BxN	Complete protection from disease development in BLT1−/− mice. Adoptive transfer of WT neutrophils is sufficient for disease development in BLT1−/− mice	(39)

Recent data has renewed the interest in the LTB₄ pathway as a target in arthritis. Chen et al., have shown that neither 5-LO nor LTA₄ hydrolase knock-out mice develop disease in the K/BxN model, while LTC₄ synthase knock-out mice develop full disease. Prophylactic treatment with a 5-LO antagonist also completely blocked disease from occurring in wild-type mice. They went on to show in mast cell and neutrophil transfer experiments that neutrophils but not the mast cells are the primary source ofLTB₄ in K/BxN arthritis [19]. Using the BLT1 knock-out animals, Luster’s group showed a critical role for BLT1 in K/BxN arthritis [37]. BLT1−/− mice do not show any signs of arthritis. Adoptive transfer of neutrophils from wild-type mice restored disease in these mice but most of the recruited neutrophils in the synovium are BLT1 negative suggesting that BLT1 is required only for initiation of joint inflammation but not maintenance. CP-105,696 was also shown to block disease occurrence in this model [37]. Our studies with BLT1 and BLT1/BLT2 deficient mice in a CIA model also implicated a critical role for BLT1 in arthritis. Both these mice showed similar levels of anti collagen antibody levels indicating normal immune response to collagen but complete absence of joint inflammation [29]. Although BLT1/BLT2 double deficient mice are protected from disease in this model, complete protection of BLT1−/− mice does not allow for determining the independent functions of BLT2 in arthritis. Generation of BLT2−/− mice is critical for identification of its role, if any, in arthritis.

Based on the current knowledge, potential mechanisms involved in arthritis development for LTB₄ and its receptors are shown in Figure 1. Arthritis might be initiated with the formation of auto-antibodies and deposition of immune complexes (IC) by as yet undefined mechanisms (reviewed in [38]). While the sequence of events is not clearly established studies have implicated macrophages, neutrophils, mast cells, dendritic cells and T-cells in different models of arthritis. All of these cells have recently been shown to express LTB₄ receptors and many cells also produce LTB₄. Amplification loops between key cytokines involved in arthritis such as TNFα, IL-1β and IL-18 to LTB₄ production have been reported. In addition, studies have also shown that C5a mediated neutrophil recruitment requires BLT1 in a mouse model of peritonitis [39]. However, a cause and effect relationship between LTB₄ responses of these cells in development of arthritis remains to be established.

Critical Questions about Leukotriene B₄ Receptors in Rheumatoid Arthritis

Several important questions remain unanswered regarding the role of LTB₄ receptors in RA. Pharmaceutical companies studied the involvement of LTB₄ in RA for nearly 25 years, yet no clear candidate drugs emerged from these studies. The discovery of BLT2 with distinct antagonist specificity and tissue distribution may provide a clue to the ineffectiveness of some of the earlier antagonists [40]. It is not surprising that these antagonists did not work well on BLT2 since they were all selected and refined using assays based on neutrophil activation by BLT1. What is the role of BLT2 in inflammatory diseases? It could have a complementary and/or compensatory role during the development of human RA. Thus, blocking one of these receptors might be ineffective for RA treatment. A specific role for BLT2 in art hritis and other inflammatory diseases remains to be established.

List of abbreviations

LTB₄

leukotriene B₄

CIA

collagen-induced arthritis

TNF

tumour necrosis factor

RA

rheumatoid arthritis

COX-2

cyclooxygenase-2

GPCR

G-protein coupled receptor

CFA

complete Freund’s adjuvant

IFA

incomplete Freund’s adjuvant

5-LO

5-lipoxygenase

LTA₄H

leukotriene A₄ hydrolase

LTA₄

leukotriene A₄

LTC₄

leukotriene C₄

FLAP

Five-Lipoxygenase Acivating Protein