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(Journal of Leukocyte Biology. 2001;70:708-714.)
© 2001 by Society for Leukocyte Biology

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex–mediated arthritis

P.L.E.M. van Lent*, R. Licht{dagger}, H. Dijkman, A.E.M. Holthuysen*, J.H.M. Berden and W.B. van den Berg

Division of Rheumatology, University Medical Centre Nijmegen, the Netherlands,
* Division of Nephrology, University Medical Centre Nijmegen, the Netherlands,
{dagger} Division of Pathology, University Medical Centre Nijmegen, the Netherlands

Correspondence: P.L.E.M. van Lent, Division of Rheumatology, University Medical Center Nijmegen, Geert Grooteplein 26-28, 6500 HB Nijmegen, the Netherlands. E-mail: p.vanlent{at}reuma.azn.nl.


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ABSTRACT
 
Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex–mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.

Key Words: joint inflammation • immune complexes • synovium • apoptotic cells • synovial lining macrophages • chemotaxis


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INTRODUCTION
 
During arthritis, large numbers of inflammatory cells migrate into the joint cavity [1 ]. Many of them undergo apoptosis, a process that follows programmed pathways leading to well-defined biochemical and morphological characteristics [2 , 3 ]. Characteristic features of apoptosis are DNA digestion in the internucleosomal linker regions, nuclear fragmentation, aggregation of dense masses of chromatin beneath the nuclear membrane, and cell shrinkage. During apoptosis, the cells’ surfaces are altered, which leads to their removal by phagocytosis [4 ]. Macrophages can identify a cell as apoptotic, and they have been shown to phagocytose apoptotic cells. Many receptors may be important in the interaction between apoptotic cells and macrophages. On the macrophage surface, the vitronectin receptor {alpha}vß3 [5 ], CD36 [6 ], the ATP-binding cassette transporter ABC-1 [7 ], class A scavenger receptor [8 ], CD14 [9 ], and possible low-density lipoprotein receptor CD68 [10 ] have been involved in apoptotic cell clearance. On the apoptotic cell, however, only one surface change—the exposure of phosphatidylserine—has been firmly linked with the clearance mechanism [11 ], whereas recently it was found that ICAM-3 (CD50) is also important in the recognition of apoptotic leukocytes by macrophages [12 ].

Both apoptosis and the removal of apoptotic cells are essential to prevent leakage of potentially harmful intracellular contents into the micro-environment [13 , 14 ]. In contrast to complement or Fc-mediated phagocytosis, the uptake of apoptotic cells does not promote inflammation [15 ]. In fact, recent data even indicate that the phagocytosis of apoptotic material by macrophages causes the release of mediators that actively inhibit the inflammatory response [16 17 18 19 ].

Di-arthrodial joints are covered by a thin layer of cells, the synovial intima [20 ]. In the mouse, the synovial intima consists mainly of macrophage-like type A cells. Recent studies have shown that these cells are of utmost importance for the onset [21 22 ], propagation [23 ], and exacerbation [24 ] of experimental arthritis. They probably form an important source of chemokines, which are released on activation by cytokines such as interleukin-1 [22 ].

Earlier we developed a model of immune complex–mediated arthritis (ICA) in the mouse. Arthritis is induced by local injection of the antigen lysozyme into the knee joint of mice that previously were given specific antilysozyme antibodies intravenously [25 ]. Maximal knee joint inflammation is found at day 2. At that stage, many inflammatory cells undergo apoptosis in the knee joint cavity. Apoptotic cells can be removed by polymorphonuclear cells present in the joint cavity or by synovial lining macrophages. Inhibition of synovial macrophages due to apoptotic cell uptake may have implications for the onset and propagation of joint inflammation.

In this study, we investigated whether apoptotic leukocytes can be phagocytosed by synovial lining cells during experimental ICA, whether macrophages that have phagocytosed apoptotic cells modulate the chemotactic activity in vitro, and the impact of apoptotic cell uptake by SLMs on the onset of experimental arthritis. Our study indicates that apoptotic cells are phagocytosed by synovial lining macrophages and that this leads to inhibition of the cell influx into the joint cavity in which experimental arthritis is subsequently elicited.


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MATERIALS AND METHODS
 
Animals
Female inbred BALB/c mice younger than 15 weeks were used for all experiments. The mice were purchased from Harlan (Horst, the Netherlands) and housed under specific pathogen-free conditions. The animals were fed standard food (Hope Farms, Woerden, the Netherlands) and acidified tap water ad libitum and treated according to university guidelines.

Induction of ICA
ICA was induced as described earlier [25 ]. Three micrograms of Henn Egg Lysozyme coupled to poly-L-lysine was injected into the knee joints of mice that had previously been given 0.2 mL antilysozyme antibodies intravenously. The first signs of arthritis were observed at 6 h, and maximal inflammation was found at day 2 after arthritis induction.

Light microscopy
Knee joints were fixed in buffered formalin, decalcified in formic acid, and embedded in paraffin. Whole knee joint sections (6 {theta}m) were cut on a Reichert microtome, mounted on gelatin-coated slides, and stained with hematoxylin/eosin as described earlier [26 ].

Electron microscopy
Synovia were dissected from the knee joint and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 2 h. Subsequently, the synovia were washed in cacodylate buffer, postfixed in 1% OsO4 for 1 h, and washed and dehydrated in increasing concentrations of ethanol. The 100% ethanol was replaced by 1,2 propylene-oxide and subsequently embedded in epon 812. Sections (90 nM) were cut with a Reichert microtome and stained with uranyl acetate and lead citrate. The sections were studied with an electron microscope (Jeol 1200EX, Japan).

Isolation of thymocytes and apoptosis induction
After cervical dislocation, the thymus of a BALB/c mouse was removed and stored in 10% fetal bovine serum (FBS) on ice. Apoptosis was induced as described [27 ]. Briefly, a single-cell suspension of thymocytes was seeded in 10% FBS in Dulbecco’s modified Eagle’s medium (DMEM) (10% FBS/DMEM) supplemented with 1 {theta}m dexamethasone (Genfarma, Maarssen, the Netherlands). After 3 h of incubation, the cells were washed 3 times in medium and resuspended in 1 mL of 10% FBS/DMEM. Apoptosis was determined by annexin-V-FITC/propidium iodide double staining [28 , 29 ], which was analyzed in a Coulter EpicsXL flow cytometer (Coulter Corporation, Hialiah, FL) equipped with a 488-nM Argon laser.

Isolation of resident peritoneal macrophages
Resident peritoneal macrophages were isolated as previously described [27 ]. Briefly, closed peritoneal lavage was performed on non-anesthetized mice. The peritoneal lavage was kept on ice until seeding on plastic coverslips in 10% FBS/DMEM and allowed to settle and adhere for 1.5 h, after which nonadherent cells were removed by washing with 10% FBS.

Macrophage and apoptotic cell interaction in vitro
In vitro phagocytosis and subsequent scoring were performed as described previously [26 ]. In short, macrophages and apoptotic thymocytes were allowed to interact for 10 min or 1 h, after which the coverslips were rinsed with 10% FBS/DMEM, using a Pasteur pipette to remove nonphagocytosed thymocytes. Immediately after interaction, coverslips were spin-dried, fixed, and stained. Preparations were scored at 400x magnification by regular light microscopy.

Determination of chemotactic activity
Isolation of PMNs
Murine neutrophil granulocytes (PMNs) were isolated after implantation of gelatin sponges (Willospon; Will-Pharma, Zwanenburg, the Netherlands), a method developed by Middleton and Campbell [30 ]. After cells were collected, analysis of the cytospin products showed that the cell suspension consisted predominantly of neutrophil granulocytes (99%).

Chemotactic assay
PMN chemotaxis was measured using 12-well 6.5-mm Costar Transwell culture plate inserts equipped with polycarbonate filters (no. 3415, pore size 3 µm, polyvinyl pyrrolidone free; Costar, Cambridge, MA). Next, 105 peritoneal macrophages were attached to coverslips and 5 x 105 apoptotic cells or non-apoptotic control cells were added and incubated for 1 h. Non-ingested apoptotic or intact thymocytes were washed away by rinsing the coverslips in 37oC DMEM. Macrophages were activated by lipopolysaccharides (LPSs) (5 µg/ml) for 5 h. Then 500 µL of culture supernatant was added to the bottom chamber of the transwell. Subsequently, 100 µL of PMN suspension (105 cells/mL) was loaded above the filter and incubated for 2 h at 37oC in humified 5% CO2. As a positive control, 5% zymosan-activated serum was used. Fresh serum was activated by 10% zymosan for 1 h at 37oC. Phosphate-buffered saline (PBS) was used as a negative control. Thereafter, cells in the bottom chamber were counted by using a hemocytometer. We also made a cytocentrifuge of 100 µL of supernatant. PMNs were stained with Giemsa-May-Grünwald and counted under the microscope. Results are expressed as the total number of cells in the bottom chamber 2 h after incubation.

Injection of apoptotic cells into murine knee joints
Apoptotic (AN+/PI-) cells were injected (2 x 106 /6 {theta}L 10% FBS/DMEM) into the knee joint of BALB/c mice. Apoptotic cells were injected in normal knee joints. At several time points (10, 30, and 60 min) after injection of apoptotic cells, mice were sacrificed and total knee joints or synovial specimens were processed by light and electron microscopy. Furthermore, apoptotic cells were injected in knee joints 1 h before arthritis induction. Total knee joint sections were stained with hematoxylin/eosin or immunostained with NIMP-R14 (an antibody that specifically binds a 25–30-kD epitope mainly found on PMNs [31 ]).

Briefly, sections were deparaffinized and pre-incubated for 15 min at RT with 20% normal rabbit serum. Thereafter, sections were incubated with NIMP-R14 antibodies for 1 h. After incubation with the second antibody rabbit anti-rat peroxidase for 30 min, the sections were stained for hematoxylin for 30 s. As a negative control, sections were incubated with normal rat IgG instead of NIMP-R14. Joint inflammation was scored as the total amount of PMNs found in synovium (infiltrate) and knee joint cavity (exsudate) using an arbitrary scale of 0–3 (0=no PMNs; 1=few; 2=some; 3=many).


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RESULTS
 
Uptake of apoptotic cells by synovial lining macrophages during early experimental ICA
To investigate whether the uptake of early apoptotic cells by synovial lining cells takes place, we first checked the occurrence of significant uptake of apoptotic leukocytes by synovial intimal cells during experimental arthritis. In our model, large amounts of leukocytes (mainly PMNs) migrated into the joint cavity and maximal inflammation was found at day 2. After ICA induction, many inflammatory cells were observed in close association with the synovial lining (Fig. 1A ). We used electron microscopy for more detailed analysis. We were able to demonstrate clear uptake of apoptotic cells in synovial macrophages 1 and 2 days after ICA induction (Fig. 1B) .



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  		Figure 1. Murine knee joints were isolated 48 h after induction of immune complex–mediated arthritis. Prominent binding and uptake of leukocytes by synovial lining macrophages (SLMs) in knee joint sections stained with hematoxylin and eosin (A) was found by light microscopy. The upper half of (A) represents the joint cavity infiltrated mainly with PMN, whereas the lower half shows the synovial layer with the thin lining layer containing predominantly type A macrophages (see arrows). The electron micrograph shows binding of PMNs and phagocytosis of an apoptotic PMN (Fig. 2B) . Uptake was only found in synovial lining macrophages. JC = joint cavity, SM = synovial macrophages, L = leukocyte, S = synovium, AC = apoptotic cell. Original magnification of light micrographs x200 and electron micrographs x6750.

Uptake of apoptotic cells by macrophages in vitro inhibits chemotactic activity for PMNs
To obtain a model system to analyze the impact of phagocytosis of apoptotic cells, apoptotic thymocytes (AN+/PI- cells) were incubated with murine peritoneal macrophages attached to plastic coverslips. AN+/PI- cells did bind and then were rapidly taken up by residential macrophages within 10 min of incubation. After 60 min of incubation, 90% of the macrophages had taken up apoptotic thymocytes (Fig. 2 ). Staining of these thymocytes with annexin V and PI showed that mostly AN+/PI- were taken up by peritoneal macrophages whereas AN-/PI- cells stayed outside the macrophage (data not shown).



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  		Figure 2. Uptake of early apoptotic cells by murine peritoneal macrophages attached to coverslips. Coverslips were fixed 60 min after the addition of apoptotic cells and were stained with hematoxylin/eosin. Original magnification x1000.

To directly investigate whether the uptake of apoptotic cells has consequences for chemotaxis for PMNs, the supernatant of macrophages given apoptotic cells or intact cells and subsequently activated by LPS for 5 h was tested for its ability to attract PMN in a chemotactic assay. Within 2 h, PMN migrated through the filter from the upper to the lower compartment, which contained the supernatant of activated macrophages. Migration of PMN induced by the supernatant of macrophages that had phagocytosed apoptotic cells was significantly lower (58%) (Fig. 3 ).



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  		Figure 3. Migration of purified mouse PMNs in the transwell chemotaxis assay. Significantly reduced migration of PMN was seen to culture supernatants of LPS-activated macrophages that previously had engulfed early apoptotic cells as compared to LPS-activated control macrophages. As a positive control, 5% zymosan-activated serum (ZAS)/DMEM was used. We used DMEM as a negative control. Values represent the mean ±SD of six measurements. data were evaluated with the mann-whitney u test (**P<0.05).

Uptake of apoptotic cells by macrophages in vivo
We further investigated whether apoptotic (AN+/PI-) cells were also phagocytosed in vivo. Synovial lining type A macrophages differ from other tissue macrophages. Type A cells are surrounded by a collagen type IV matrix, which may disturb an efficient uptake. AN+/PI- or intact cells (AN+/PI-) were injected directly into the knee joint (2 x 106 in 6 µL).

Histological examination of total knee joints at 10, 20, and 30 min after injection of intact AN+/PI- cells showed no binding and uptake of these cells by synovial lining cells (data not shown). In contrast, a clear binding to and uptake of AN+/PI- apoptotic cells was found (Fig. 4A ). About 10% of the cells injected in the joint cavity bound to the lining layer 10 min after injection. The binding decreased thereafter. Electron microscopy identified type A cells, characterized by large cell protrusions, as the cell type that phagocytosed apoptotic cells (Fig. 4B) . Type B cells, which have a more fibroblastic appearance, did not bind or phagocytose apoptotic cells.



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  		Figure 4. Light (A) and electron micrographs (B) of knee joints, shortly after injection of apoptotic cells directly into the knee joint (2 x 106 apoptotic cells in 6 {theta}l 10% FBS/DMEM). In the knee joints, apoptotic cells were ungulfed by type A lining macrophages covering the inside of the knee joints 10 min after intra-articular injection. SM = synovial macrophage, AC = apoptotic cell. Magnification light micrograph (A) = x1000 and electron micrograph (B) = x9700.

Uptake of apoptotic cells by synovial lining macrophages inhibits the onset of ICA
To investigate whether lining cells that have phagocytosed apoptotic cells influence the onset of ICA, 2 x 106 apoptotic cells or intact control cells were injected directly into the knee joint 1 h before arthritis induction. At 6 h and 24 h after induction, mice were sacrificed and total knee joints were processed for histology. In addition, total knee joints were either stained with hematoxylin or immunostained with NIMP-R14, an antibody that specifically stains PMNs. This allowed us to score the amount of PMN in infiltrate and exsudate using an arbitrary scale. We found that the infiltrate in the synovial layer was the same at 6 and 24 h among the groups that received either apoptotic cells or intact control cells (Fig. 5A ). The amount of PMNs in the exsudate, however, was significantly lower (61%) in the joints pretreated with apoptotic cells at 24 h but not at 6 h after ICA induction when compared to controls (Fig. 5A 5B 5C) .



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  		Figure 5. Cell influx into the joint cavity (exudate) or synovium (infiltrate) in arthritic knee joint sections stained with hematoxylin and eosin was scored on a 0–3 scale (0=no influx, 1=minor, 2=moderate, 3=marked). One hour before arthritis induction, 2 x 106 apoptotic cells in 6 µL 10% FBS/DMEM were injected into the knee joints. Note the significant reduction in cell influx in the exudate at 24 h but not at 6 h after arthritis induction (A and micrographs B = intact cells injected versus C = apoptotic cell injected). No differences were found in the infiltrate both at 6 and 24 h. Values represent 2 experiments with 5 mice per group. *P < 0.05 versus PBS alone by Mann-Whitney U test. P = patella, F = femur, JS = joint space, S = synovium. Original magnification of photographs x400.


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DISCUSSION
 
In this study we find clear uptake of apoptotic cells by synovial lining macrophages during ICA. Apoptotic cells are taken up by almost every cell type in the body, but macrophages have been very efficient in clearing these cells. During rheumatoid arthritis (RA), prominent apoptosis occurs within the synovial tissue. Using in situ end labeling and agarose gel electrophoresis detecting DNA strand breaks, it was found that the primary localization of the apoptotic cells was within the synovial lining macrophages [32 ]. Another study showed a close correlation between the increasing propensity of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population [33 ].

First we studied the effect of apoptotic cell uptake by macrophages on chemotactic activity for PMN. Using a transwell chemotactic assay for PMNs, we found that chemotactic activity produced by activated macrophages was significantly decreased when they had engulfed apoptotic cells. Dexamethasone used for induction of apoptotis in thymocytes may have an effect on macrophage function. However, the dexamethasone concentration measured in the apoptotic cell suspension was lower than 10-11 mM, and this concentration had no effect on cytokine production (IL-1ß, TNF{alpha}) by macrophages after stimulation by LPS (data not shown). Earlier studies revealed that apoptotic cell uptake by mouse J744 macrophages inhibited synthesis and secretion of the chemokines’ macrophage inflammatory protein-2 (MIP-2) and macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), which predominantly attract PMNs. The predominantly monocyte-attracting chemokine monocyte chemotactic protein (MCP-1/JE), however, was not inhibited [18 ]. Furthermore, the production of other mediators such as IL-1 [34 ] and enzymes [35 ] was decreased. Inhibition of chemokine production is probably regulated by TGF-ß since this growth factor was increased [18 , 19 , 36 ], whereas anti-TGF-ß neutralizing antibodies reversed the inhibiting effect of apoptotic cell uptake [34 ]. Moreover, TGF-ß is released by human macrophages in response to ingestion of apoptotic neutrophils [34 ], whereas exogenous TGF-ß down-regulated the synthesis of the PMN-specific chemokine production [18 ].

To determine whether this in vitro inhibitory effect was also found in vivo, we first investigated whether early apoptotic cells are engulfed by type A intimal cells if injected into the joint. Type A intimal cells lie within a reticular network that is presumably a product of type B synovial cells and consists of collagen type IV, heparan sulfate proteoglycan, and laminin [37 ]. Despite the layer surrounding the synovial type A cells, we found prominent binding and uptake of apoptotic cells at 10 min after intra-articular injection. More than 18 distinct integrins have been described on the synovial macrophage that may be involved in this process [37 ]. During joint inflammation, the components of the reticular network surrounding the macrophage are partly degraded by enzymes, which may thus lead to exposure of receptors that are more efficiently involved in clearance of apoptotic cells.

We further analyzed whether apoptotic cell uptake by type A cells had consequences for joint inflammation. When injected into murine knee joints before experimental arthritis induction, apoptotic cells caused inhibition of PMN influx within the joint cavity. In the immune complex arthritis model, two chemotactic factors seem of utmost importance for PMN migration: namely, C5a and IL-1. Elimination of C5a by Cobra venom factor pretreatment before arthritis induction prevented PMN migration for the most part [25 ]. C5a probably forms the main PMN-attracting component during the first hours of this arthritis. Since we found no effect of apoptotic cell uptake by synovial macrophages on arthritis development after 6 h, we concluded that apoptotic cells probably do not alter C5a generation. The second major regulating factor is IL-1, which is formed several hours after the onset of arthritis [25 ]. Neutralization of the formed IL-1 using anti-IL-1 antibodies [25 ] or IL-1ra [38 ] largely prevented the influx of PMNs. IL-1 is not chemotactic but probably acts by mediating the production of PMN-attractive factors such as MIP-2 and MIP-1{alpha} by synovial macrophages. Engulfment of apoptotic cells by macrophages may thus inhibit synthesis and secretion of these PMN-attracting chemokines either directly or indirectly by lowering IL-1 production. Synovial lining macrophages were important producers of chemokines in this model since selective removal of these cells using clodronate-containing liposomes before arthritis induction prevented both joint inflammation and chemokine production [39 ].

Engulfment of apoptotic cells by lining macrophages significantly inhibited PMN migration into the joint cavity but not in the synovial layer 24 h after arthritis induction. In earlier studies we found that complete removal of type A cells before ICA induction completely blocked synovial and joint cavity cell influx [39 ]. It seems likely that apoptotic cell engulfment blocks superficial lining macrophage activation only in part, because the local chemokine levels are sufficient to attract inflammatory cells into the synovium but insufficient to promote traffic to the joint space. When other particles, such as empty liposomes, were injected, they were also selectively taken up by lining macrophages, but this did not alter the onset and severity of ICA [21 ], which means that the uptake of particles alone does not lower the inflammatory function of the lining macrophage.

During RA, large amounts of inflammatory cells are found within the synovial fluid. These cells posses potent degrading enzymes and pro-inflammatory mediators, and their removal is vital for resolution of the inflammation. Apoptosis and subsequent disposal by macrophages have been a major route for direct removal of effete inflammatory cells. This study shows that apoptotic cells are engulfed by resident synovial lining macrophages, which are crucial in the onset, propagation, and exacerbation of arthritis. Inhibition of residential synovial macrophages lining the joint cavity, by uptake of apoptotic cells, may be an important physiological mechanism contributing to the resolution of joint inflammation.

Received March 17, 2001; revised July 1, 2001; accepted July 9, 2001.


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