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Pathology International 2014; 64: 543–550
doi:10.1111/pin.12208
Original Article
Role of blood ribosomal protein S19 in coagulum resorption: A study using Gln137Glu-ribosomal protein S19 gene knock-in mouse
Jun Chen,1 Rika Fujino,1 Rui Zhao,1 Umeko Semba,1 Kimi Araki2 and Tetsuro Yamamoto1 1
Department of Molecular Pathology, Faculty of Life Science and Graduate School of Medical Sciences and Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan 2
Sera of human, guinea pig or mouse contain a strong monocyte chemoattractant capacity that is attributed to the ribosomal protein S19 (RP S19) oligomers generated during blood coagulation. In contrast, sera prepared from Gln137Glu-RP S19 gene knock-in mice contained negligible chemoattractant capacity. When coagula that had been preformed from the blood of both the wild type and knock-in mice were intraperitoneally inserted into host mice, after 3 days of recovery, the knock-in mouse coagula remained larger than the wild type mouse coagula. The wild type mouse coagula were covered by multiple macrophage layers at the surface and were infiltrated inside by macrophages. Knock-in mouse coagula exhibited less macrophage involvement. When coagula of knock-in mice and coagula of knock-in mice containing C5a/RP S19, an artificial substitute of the RP S19 oligomers, were intraperitoneally inserted as pairs, the C5a/RP S19 containing coagulum was more rapidly absorbed, concomitant with increased macrophage involvement. Finally, when the knock-in mouse and wild type mouse coagula pairs were inserted into mice in which macrophages had been depleted using clodronate liposome, the size difference of recovered coagula was reversed. These results indicate the importance of the RP S19 oligomer-induced macrophage recruitment in coagulum resorption. Key words: clodronate liposome, coagulum resorption, extraribosomal function, knock-in mouse, macrophage recruitment, ribosomal protein S19
Correspondence: Tetsuro Yamamoto, MD, PhD, Laboratory Testing Management Section, Saiseikai Kumamoto Hospital, 5-3-1 Chikami, Minami-ku, Kumamoto 861-4193, Japan. Email: tetsuro [email protected] Jun Chen and Rika Fujino contributed equally to this work. Disclosure: The authors have no financial interest in any of the products and methods described in this manuscript. Received 14 March 2014. Accepted for publication 2 September 2014. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Ribosomal Protein S19 (RP S19) is a component of the ribosomal small subunit that is reported to be essential in ribosome biogenesis.1 Interestingly, RP S19 is also present in normal blood plasma in complex with prothrombin.2,3 During blood coagulation, RP S19 is oligomerized by activated factor XIIIa, a transglutaminase that catalyzes the formation of an intermolecular isopeptide bond between Gln137 and Lys122.4 Upon this intermolecular cross-linkage, the RP S19 oligomers gain the extra-ribosomal function of monocyte/ macrophage-selective chemoattraction.5 This results in the generation of monocyte chemotactic capacity in serum in vitro.2,6,7 RP S19 oligomers, but not monomers, exhibit monocyte chemoattraction by acting as a ligand for the C5a receptor.5 Monocyte/macrophage-selective recruitment is provided by the dual effects of RP S19 oligomers as agonists of the C5a receptor of monocytes/macrophages and antagonists of the C5a receptor of neutrophils.5 The neutrophil-selective antagonist effect is attributed to the C-terminal 12 amino acid residues of RP S19,8,9 and a recombinant chimeric protein C5a/RP S19, in which the RP S19 C-terminal 12 residues are connected to the C-terminal of C5a, reproduces the dual effects of the RP S19 oligomers.10 We previously developed the coagulum absorption model in the peritoneal cavity of guinea pig to examine the biological role of the RP S19 oligomers generated in the blood coagulum. After intra-peritoneal transplantation, the coagulum is covered and infiltrated by macrophages within a day, and coagulum components are engulfed by the infiltrated macrophages.11 Currently, we prepared a homozygous gene knock-in mouse in which the RP S19 gene was replaced by a Gln137Glu-RP S19 artificial gene. The Gln137Glu mutation seems to cause dysfunction specific to the extra-ribosomal function of RP S19. In the current study, we have reexamined the role of RP S19 in blood plasma using the knock-in mouse-derived materials.
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Importance of macrophages in biological events was sometimes shown by the macrophage-depletion experiment. A technique to deplete macrophages from mice using liposome encapsulating clodronate (dichloromethylenebisphosphonate) which is an ATP mimetic reagent has been established,12 and the clodronate liposome is now commercially available. In the current study, we used clodronate liposome-treated mice to demonstrate the importance of macrophages in the intraperitoneal coagulum resorption model.
C57BL/6J and intercrossed the heterozygous Gln137GluRP S19 knock-in mice to obtain homozygous mice. We confirmed homozygous Gln137Glu-RP S19 knock-in by PCRSouthern blotting analysis. The homozygous knock-in mouse was fertile and apparently grew normally. The wild type and knock-in mice were maintained in the Center for Animal Resources and Development, Kumamoto University. The animal experiments were performed under the control of the Ethical Committee for Animal Experiment, Kumamoto University School of Medicine (approval number: B23-165).
Monocyte chemotaxis assay
MATERIALS AND METHODS Proteins and reagents We obtained rabbit IgG conjugated with fluorescein isothiocyanate (FITC) from Sigma-Aldrich (St. Louis, MO). We prepared recombinant C5a and recombinant chimeric protein C5a/RP S19 as described previously.10 We purchased all other chemicals from Nacalai Tesque (Kyoto, Japan) or from Wako Pure Chemicals (Osaka, Japan) unless otherwise specified.
Preparation of Gln137Glu-RP S19 knock-in homozygous mouse bearing C57BL/6J background We prepared Gln137Glu-RP S19 knock-in homozygous mice bearing a C57BL/6J genetic background. We purchased specific pathogen-free Crij:CD1 (ICR) and C57BL/6J mice (15– 20 g body weight range) from Charles River (Yokohama, Japan). We utilized a BAC#24 158H15 clone (BACPAC Resources Children’s Hospital Oakland, Oakland, CA, USA) bearing mouse RP S19 exon 5, which codes for Gln137. Using this clone we prepared Gln(CAG)137Glu(GAG)mutated exon 5 by means of polymerase chain reaction (PCR), and the PCR product was inserted into pGEM-T Easy vector (Promega, Tokyo, Japan). We replaced wild type exon 5 in a RP S19 gene fragment in pBS vector by the mutant exon 5 with a marker cassette (Lox71-SA-IRES-neomysinpA-Lox71).13 After amplification of the mutant exon 5 in host DH5α cells, we cut off the mutated RP S19 gene fragment with restriction enzyme SacI. We homologously replaced the mutated RP S19 gene fragment to the same position as the wild type RP S19 gene of embryonic stem cells of TT2KTPU8 F1 mice using electroporation. We implanted the mutant RP S19 embryonic stem cells along with germinal cells of ICR mice into endometrium of female mice and mated the ICR mice progeny exhibiting 100% chimerism with Cre C57BL/6J transgenic mice to remove the marker cassette.13 We repeatedly backcrossed the knock-in heterozygous mice with wild type mice to adjust the genetic background to
We obtained peripheral blood into a heparinized syringe from healthy volunteers after getting their informed consent under approval of Institutional Review Board of Kumamoto University (approval number 673). We isolated mononuclear cells from the heparinized blood using Ficoll-Paque Plus (Amersham Biosciences KK, Tokyo, Japan). The mononuclear cell fraction contained approximately 20% monocytes when identified as the macrophage specific esterase positive cells.9 We evaluated the monocyte-attracting capacity of mouse sera using a 48-well chemotaxis chamber with a polycarbonate filter (5 μm pore size) (Neuro Probe, Inc., Gaithersburg, MD, USA) according to the method of Folk et al.14 After incubation for 90 min, we stained the membrane with Giemsa solution and counted the total number of monocytes that migrated beyond the lower surface of the membrane in five high-power microscopic fields. We have previously described these methods in detail.15
Preparation of mouse blood and serum We obtained mouse blood from retro-orbital plexus of wild type C57BL/6J mice or the knock-in mice with 27 gauge needles and used to prepare coagulum and serum. To prepare serum, we allowed blood to spontaneously coagulate in sterilized glass tubes for 60 min at 22°C, followed by centrifugation at 4000 × g for 20 min at 22°C.
Intraperitoneal coagulum resorption model To prepare mouse blood coagulum, we initially mixed 10 μL of FITC-conjugated rabbit IgG in phosphate-buffered saline (PBS) as a marker (final concentration 2 mg/mL, SigmaAldrich, St. Louis, MO, USA), C5a/RP S19 in PBS (final concentration 10−8 M) or PBS alone with 100 μL aliquots of liquid blood. We then poured the mixture into a glass cylinder that was covered on one end with a sterilized parafilm sheet to form the coagulum. We performed a small midline lapa-
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rotomy on C57BL/6J mice (wild type and the knock-in) under ether anesthesia, inserted two coagula, sutured the abdominal incision, and provided water and food ad libitum for 3 days prior to performing a surgical operation to recover the coagulum. After recovery, coagula were weighed and fixed in 10% formalin. We have previously reported this method using guinea pigs in detail.11
Preparation of clodronate liposome-treated mice We attempted to deplete macrophages according to the method of van Rooijen and Sanders.12 We used liposome encapsulating clodronate and control liposome produced by Hygieia Bioscience (Minoo, Japan). We diluted an aliquot of each liposome (10 mg/mL) 10 fold with PBS upon the treatment of mice. We intraperitoneally injected with 200 μL of one of the working liposome suspensions into each mouse (10 mg liposome/kg body weight) once a day for 3 continuous days. Next day, we subjected the mice to the intraperitoneal coagulum insertion experiment. Because we often observed paralysis of hind extremities in clodronate liposome-treated male mice, we only used female mice for the clodronate liposome treatment.
Histological examination After being weighed, the coagula recovered from the peritonea were immediately fixed in neutralized 10% formalin for preparation of paraffin sections. Paraffin sections with 4 μm thickness were stained with hematoxylin and eosin in the usual way. The histologic specimens were observed under a microscope, Provis AX (Olympus, Tokyo, Japan) with a digital camera, Penguin 600CL (Pixera, Los Gatos, CA, USA).
Figure 1 Absence of monocyte chemotactic capacity in serum of Gln137Glu-ribosomal protein S19 (RP S19) gene knock-in mouse. Chemotaxis chamber assay was performed for mouse sera prepared from retro-orbital plexus blood of a wild type mouse (WT-serum) and of a Gln137Glu-RP S19 knock-in mouse (KI-serum) using human peripheral blood monocytes. Upon the chemotaxis assays the samples were diluted 10 fold (10), 100 fold (102) or 1000 fold (103) with phosphate-buffered saline (PBS) and used (open columns). As the negative and positive controls, PBS and a recombinant human complement C5a (C5a) at 10−8 M were respectively used (closed columns). After a 90-min incubation of the micro-well chamber, the chemotaxis membrane was stained with Giemsa solution and the total number of monocytes migrated beyond the membrane was counted in five microscopic high-power fields. Each sample was evaluated in micro-wells, and the total number of migrated monocytes is shown as the mean ± standard deviation. Results with 10 fold diluted samples were analyzed with the paired Student’s t-test (P = 0.001). We repeated the same experiments using 3 pairs of wild type mice and the knock-in mice. The results were reproducible and data of a representative experiment are shown.
Statistical analysis
capacity. Serum prepared from peripheral blood of wild type mice possessed monocyte chemoattractant capacity, as observed in human and guinea pig sera.2,6,7,11 In contrast, serum prepared from Gln137Glu-RP S19 knock-in mice did not exhibit monocyte chemoattraction at all (Fig. 1).
We performed statistical analyses using unpaired or paired Student’s t-tests. We considered P ≤ 0.05 to be statistically significant (*P ≤ 0.05, **P ≤ 0.01).
Delay of resorption of Gln137Glu-RP S19 knock-in mouse-derived coagulum in peritoneum
RESULTS Lack of monocyte chemotactic capacity in Gln137Glu-RP S19 knock-in mouse-derived serum During blood coagulation, RP S19 monomers in plasma are cross-linked by the factor XIIIa-catalyzed reaction, becoming homo-oligomers and gaining monocyte chemoattraction
We next performed the coagulum resorption experiment in the mouse peritoneal cavity. To control the effects of variability among animals, we inserted a pair of coagula intraperitoneally into wild type and knock-in mice. For an inserted pair, one coagulum was prepared from wild type mouse blood, and the other was from knock-in mouse blood. During the preparation of the knock-in mouse coagulum, we added FITC-labeled IgG to distinguish the two in the peritoneum after recovery. In the initial experiment, we mixed FITC-
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Figure 2 Delay of resorption of Gln137Glu-RP S19 knock-in mousederived coagulum in peritoneum. One coagulum was prepared with 100 μL of peripheral blood of a wild type mouse which had been mixed with 10 μL of fluorescent isothiocyanate (FITC)-conjugated rabbit IgG as a marker in PBS (2 mg/mL at the final), the other one was from the knock-in mouse blood pre-mixed with 10 μL of PBS. The two coagula were inserted as a pair into the peritoneal cavity (a) of a wild type mouse (n = 9) or (b) of a knock-in mouse (n = 6). Three days later, the coagula were recovered, checked for the fluorescent activity, and weighed. The closed circles indicate the weight of coagula. Each two circles connected by a bar were recovered coagula pair from an individual mouse peritoneum. There are significant differences between these groups when analyzed by the paired Student’s t-test (a, P = 0.005; b, P = 0.043). To neglect unknown adverse effect of FITCconjugated IgG, we repeated the experiment of (a) but premixing FITC-IgG into the knock-in mouse coagulum. (c) The closed column and the open column indicate weights of recovered knock-in micederived coagula and wild type micederived coagula, respectively (n = 5, P = 0.01 in paired Student’s t-test). (d) Macroscopic pictures of a representative pair recovered from a wild type mouse peritoneum are shown.
conjugated IgG into the wild type mouse-derived blood. Three days after insertion, we recovered and compared the weights of the two coagula. Regardless of the genotype of the host mouse, the knock-in mouse-derived coagulum was larger than the wild type (P = 0.005 in the cases of coagula from wild type mouse peritoneum and P = 0.043 in the cases of coagula from knock-in mouse peritoneum) (Fig. 2a,b). To neglect unknown adverse effect of FITC-conjugated IgG, we repeated the experiment using peritonea of wild type mice but mixing FITC-conjugated IgG into the knock-in mousederived blood. The results were basically the same. The knock-in mouse-derived coagulum was recovered with a significantly larger size than the wild type mouse-derived coagulum again (Fig. 2c, P = 0.01). Macroscopic images of a recovered coagula set are shown in Fig. 2d.
Promotion of Gln137Glu-RP S19 knock-in mouse-derived coagulum resorption by supplementation with C5a/RP S19 To confirm that the delay of absorption of the knock-in mouse-derived coagula was due to the absence of the RP S19 oligomers, we supplemented the coagula with C5a/ RP S19. We prepared two types of knock-in mouse-derived coagula; one contained 10−8 M C5a/RP S19 and the other contained FITC-conjugated IgG. We then inserted these coagula as a pair into wild type mouse peritonea and recovered both coagula 3 days later. In all pairs, the C5a/RP S19supplemented coagulum became smaller than the control coagulum (P = 0.027) (Fig. 3a). To neglect unknown adverse effect of FITC-conjugated IgG again, we repeated the experi-
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Figure 3 Promotion of Gln137Glu-RP S19 knock-in mouse-derived coagulum resorption by supplementation with C5a/RP S19. A coagulum containing C5a/RP S19 (10−8 M at the final) which is a functional analogue of the RP S19 oligomers (KI Coagula + C5a/RP S19) and a coagulum containing FITC-IgG (KI Coagula) were inserted as a pair into the peritoneum of a wild type mouse. Three days later, the coagula were recovered, checked for the fluorescent activity, and weighed. The closed and open circles indicate the weight of coagula. Each two circles connected by a bar were recovered coagula pair from an individual mouse. (a) There is a significant difference between these groups when analyzed by the paired Student’s t-test (P = 0.027). To neglect unknown adverse effect of FITC-conjugated IgG, we repeated the experiment but premixing FITC-conjugated IgG into the C5a/RP S19-supplemented coagulum. (b) The closed column and the hatched column indicate weights of recovered knock-in mice-derived coagula with or without C5a/RP S19 supplementation, respectively (n = 5, P = 0.003 in paired Student’s t-test).
ment but premixing FITC-conjugated IgG into the C5a/ RP S19-supplemented coagulum. The results were basically the same. The C5a/RP S19-supplemented coagula recovered in significantly smaller sizes (P = 0.003) (Fig. 3b).
Histological observation of coagula recovered from peritonea We examined the recovered coagula histologically. In the case of wild type mice-derived coagula, the surface was covered by macrophages, and the inside was infiltrated by macrophages when it was transplanted into both wild type and knock-in mouse peritonea. The surface macrophage layer then became multiple and finally changed to a granulation-like structure (Fig. 4a). Infiltrating macrophages would engulf red blood cells within the coagulum as previously observed in the guinea pig coagula using an electron microscope.11 This process was greatly delayed in the case of knock-in mouse-derived coagula, regardless of the genotype of the mouse used for transplantation (Fig. 4b,d). After supplementation with C5a/RP S19, the histologic picture of the knock-in mouse-derived coagula was similar to that of the wild type-derived coagula: the surface was totally covered by multiply-layered macrophages (Fig. 4c).
Influence of clodronate liposome pretreatment of mice to coagulum resorption To confirm the important role of peritoneal macrophages, regardless the resident or the exudate, in the coagulum resorption, we tried to deplete peritoneal macrophages and precursor cells of them by pretreatment of mice with clodronate liposome. We judged the macrophage depletion from histological change of the spleen such as significant red pulp atrophy concomitant loss of macrophages (data not shown). After treatment of mice either with clodronate liposome or with control plain liposome for 3 days, we intraperitoneally inserted a pair of knock-in mouse-derived coagulum and wild type mouse-derived coagulum, and recovered the coagula 3 days later in the same way as describe above. Weights of recovered coagula are shown in Fig. 5. In the control liposome-treated group, the weights of knock-in mice-derived coagula were still heavier than those of wild type mice-derived coagula, although the size difference between the two coagula types was smaller when compared to the result with non-pretreated mice (see Fig. 2a). Distinct from this, the sizes of knock-in micederived coagula were even smaller than those of wild type mice-derived coagula in the clodronate liposome pretreated group. Histologically, a poor macrophage infiltration was
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Figure 4 Histological observation of coagula recovered from peritonea. A piece of each coagulum was fixed in neutralized 10% formalin, and a paraffin section of it was stained with hematoxylin and eosin. A pair of coagula (a) from wild type mouse blood and (b) from the knock-in mouse recovered from wild type mouse peritoneum (see Fig. 2a). A pair of coagula prepared from knock-in mouse blood premixed (c) with C5a/RP S19 or (d) with 10 μL of FITC-conjugated IgG. In c and d images, difference of the surface covering macrophage layers is focused (see Fig. 3a).
evidently observed on the coagulum surface regardless the knock-in mouse-derived coagula or the wild type mousederived coagula. Interestingly enough, focal neutrophil infiltration was observed on the surface of knock-in mousederived coagula (Fig. 6).
DISCUSSION The presence of a ribosomal protein in circulating plasma possessing an anticipative extra-ribosomal function seems unique for RP S19.2,3,7,11 We prepared Gln137Glu-RP S19 knock-in mouse in which the extra-ribosomal function was diminished but the ribosomal function was maintained. Because the knock-in mouse was fertile and apparently grew normally, the extra-ribosomal function-specific diminishment
in the knock-in mouse is suspected. This was supported at least in part by the lack of the monocyte chemoattraction capacity in the knock-in mouse-derived serum (Fig. 1). The resorption of knock-in mouse-derived coagulum in the peritoneal cavity was delayed (Fig. 2) in concomitance with a poor macrophage infiltration (Fig. 4b,d). This difference is similar to the difference between plain guinea pig coagula and coagula containing neutralizing IgG against the RP S19 oligomers after 3 days in the guinea pig peritoneum.11 Meeting our expectation, the delayed resorption of knock-in mouse-derived coagulum was reversed by supplementation of C5a/RP S19, a substitute of the RP S19 oligomers,10 in the knock-in mouse-derived coagulum (Fig. 3) accompanied by enhanced macrophage recruitment (Fig. 4c). The present data indicate again the importance of the RP S19 oligomers as macrophage chemoattractant in the coagulum resorption.
© 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Blood RP S19 in coagulum resorption
Figure 5 Influence of clodronate liposome pretreatment of mice to coagulum resorption. Wild type mice were intraperitoneally injected with 200 μL suspension of either liposome encapsulating clodronate (n = 6) (a) or control plain liposome (n = 4) (b) (10 mg liposome/kg body weight) once a day for 3 continuous days. Next day, a pair of knock-in mouse-derived coagulum and wild type mouse-derived coagulum was intraperitoneally inserted into each mouse. The marker FITC-conjugated IgG was mixed into the wild type micederived coagula in this experiment. Three days later, the coagula were recovered and weighed. The solid columns and open columns denote the weights of knock-in mice-derived coagula and the weights of wild type mice-derived coagula, respectively. Statistical analysis was performed using the paired Student’s t-test. ■, KI; □, WT.
The prompt resorption of the wild type mouse-derived coagulum was observed in the peritoneal cavity of the knock-in mouse as in the case of the wild type mouse. This suggests normal chemotactic function of macrophages in the knock-in
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mouse to the RP S19 oligomers. This has been separately confirmed in vitro (Zao et al., unpublished data). In the current study, we depleted peritoneal macrophages and their precursors by intraperitoneal injection of the clodronate liposome once a day for 3 continuous days. We had anticipated an equal delay of the resorption between the knock-in mouse-derived coagulum and the wild type-mouse derived one. However, the size of the recovered knock-in mouse-derived coagulum was even smaller (Fig. 5a). Histologically, the surface macrophage infiltration seemed equally poor between these coagula types. Focal neutrophil infiltration was observed on the surface of knock-in mouse-derived coagula in at least several sections (Fig. 6). Therefore, a possible mechanism which may explain the relatively faster resorption of the knock-in mouse-derived coagulum would be participation of neutrophils in the coagulum resorption under the absence of macrophages. The clodronate liposome treatment was reported not to affect the neutrophil number.12 In addition to this, the RP S19 oligomers antagonize the C5a receptor of neutrophils in the chemotactic response,5,8,9 and promote neutrophil apoptosis.16 These would support the less involvement of neutrophils in the RP S19 oligomer-contained wild type-derived coagulum. However, more experimental studies are required to confirm the involvement of neutrophils in the coagulum resorption especially under the absence of macrophages. The present study confirmed the importance of blood RP S19 in the macrophage-mediated resorption of coagulum. We believe that the Gln137Glu-RP S19 knock-in mouse would be a useful tool for examining the roles of blood RP S19 in the resolution of intravascular thrombotic diseases in the future.
Figure 6 Histological observation of coagula recovered from peritoneum of clodronate liposome pretreated mouse. Pairs of coagula recovered from peritonea of the clodronate liposome pretreated mice as described in Fig. 5 were fixed in 10% neutral formalin, and paraffin sections of them were stained with hematoxylin and eosin. A representative set of (a) wild type mouse-derived coagulum and (b) knock-in mouse-derived coagulum are shown. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
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ACKNOWLEGEMENTS We thank Ms T. Kubo for her technical assistance in the histological preparations. This work was supported by a Grant-in Aid for Scientific Research C (KAKENHI 24590484 to T. Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and by A-STEP (AS251Z01945Q to T.Y.) from Japan Science and Technology Agency. REFERENCES 1 Idol RA, Robledo S, Du HY et al. Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production. Blood Cell Mol Dis 2007; 39: 35–43. 2 Semba U, Chen J, Ota Y et al. A plasma protein indistinguishable from ribosomal protein S19: Conversion to a monocyte chemotactic factor by a factor XIIIa-catalyzed reaction on activated platelet membrane phosphatidylserine in association with blood coagulation. Am J Pathol 2010; 176: 1542–51. 3 Nishiura H, Tanase S, Tsujita K et al. Maintenance of ribosomal protein S19 in plasma by complex formation with prothrombin. Eur J Haematol 2011; 86: 436–41. 4 Nishiura H, Tanase S, Sibuya Y, Nishimura T, Yamamoto T. Determination of the cross-linked residues in homo-dimerization of S19 ribosomal protein concomitant with exhibition of monocyte chemotactic activity. Lab Invest 1999; 79: 915–23. 5 Nishiura H, Shibuya Y, Yamamoto T. S19 ribosomal protein cross-linked dimer causes monocyte predominant infiltration by means of molecular mimicry to complement C5a. Lab Invest 1998; 78: 1615–23. 6 Kukita I, Yamamoto T, Kawaguchi T, Kambara T. Fifth component of complement (C5)-derived high-molecular-weight macrophage chemotactic factor in normal guinea pig serum. Inflammation 1987; 11: 459–79.
7 Okamoto M, Yamamoto T, Matsubara S et al. Factor XIIIdependent generation of 5th complement component(C5)derived monocyte chemotactic factor coinciding with plasma clotting. Biochim Biophys Acta 1992; 1138: 53–61. 8 Shrestha A, Shiokawa M, Nishimura T et al. Switch moiety in agonist/antagonist dual effect of S19 ribosomal protein dimer on leukocyte chemotactic C5a receptor. Am J Pathol 2003; 162: 1381–88. 9 Nishiura H, Zhao R, Yamamoto T. The role of the ribosomal protein S19 C-terminus in altering the chemotaxis of leukocytes by causing functional differences in the C5a receptor response. J Biochem 2011; 150: 271–7. 10 Oda Y, Tokita K, Ota Y et al. Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively. J Biochem 2008; 144: 371–81. 11 Ota Y, Chen J, Shin M et al. The presence of ribosomal protein S19-like molecule in guinea pig plasma and its role in blood coagulum resorption. Exp Mol Pathol 2011; 90: 19– 29. 12 Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J Immunol Meth 1994; 174: 83–93. 13 Araki K, Araki M, Yamamura K. Targeted integration of DNA using mutant lox sites in embryonic cells. Nucleic Acids Res 1997; 25: 868–72. 14 Falk W, Goodwin RH Jr, Leonard EJ. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33: 239–47. 15 Matsubara S, Yamamoto T, Tsuruta T et al. Complement C4-derived monocyte-directed chemotaxis-inhibitory factor: A molecular mechanism to cause polymorphonuclear leukocytepredominant infiltration in rheumatoid arthritis synovial cavities. Am J Pathol 1991; 138: 1279–91. 16 Nishiura H, Zhao R, Chen J, Taniguchi K, Yamamoto T. Involvement of regional neutrophil apoptosis promotion by ribosomal protein S19 oligomers in resolution of experimental acute inflammation. Pathol Int 2013; 63: 581–90.
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Pathology International 2014; 64: 543–550
doi:10.1111/pin.12208
Original Article
Role of blood ribosomal protein S19 in coagulum resorption: A study using Gln137Glu-ribosomal protein S19 gene knock-in mouse
Jun Chen,1 Rika Fujino,1 Rui Zhao,1 Umeko Semba,1 Kimi Araki2 and Tetsuro Yamamoto1 1
Department of Molecular Pathology, Faculty of Life Science and Graduate School of Medical Sciences and Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan 2
Sera of human, guinea pig or mouse contain a strong monocyte chemoattractant capacity that is attributed to the ribosomal protein S19 (RP S19) oligomers generated during blood coagulation. In contrast, sera prepared from Gln137Glu-RP S19 gene knock-in mice contained negligible chemoattractant capacity. When coagula that had been preformed from the blood of both the wild type and knock-in mice were intraperitoneally inserted into host mice, after 3 days of recovery, the knock-in mouse coagula remained larger than the wild type mouse coagula. The wild type mouse coagula were covered by multiple macrophage layers at the surface and were infiltrated inside by macrophages. Knock-in mouse coagula exhibited less macrophage involvement. When coagula of knock-in mice and coagula of knock-in mice containing C5a/RP S19, an artificial substitute of the RP S19 oligomers, were intraperitoneally inserted as pairs, the C5a/RP S19 containing coagulum was more rapidly absorbed, concomitant with increased macrophage involvement. Finally, when the knock-in mouse and wild type mouse coagula pairs were inserted into mice in which macrophages had been depleted using clodronate liposome, the size difference of recovered coagula was reversed. These results indicate the importance of the RP S19 oligomer-induced macrophage recruitment in coagulum resorption. Key words: clodronate liposome, coagulum resorption, extraribosomal function, knock-in mouse, macrophage recruitment, ribosomal protein S19
Correspondence: Tetsuro Yamamoto, MD, PhD, Laboratory Testing Management Section, Saiseikai Kumamoto Hospital, 5-3-1 Chikami, Minami-ku, Kumamoto 861-4193, Japan. Email: tetsuro [email protected] Jun Chen and Rika Fujino contributed equally to this work. Disclosure: The authors have no financial interest in any of the products and methods described in this manuscript. Received 14 March 2014. Accepted for publication 2 September 2014. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Ribosomal Protein S19 (RP S19) is a component of the ribosomal small subunit that is reported to be essential in ribosome biogenesis.1 Interestingly, RP S19 is also present in normal blood plasma in complex with prothrombin.2,3 During blood coagulation, RP S19 is oligomerized by activated factor XIIIa, a transglutaminase that catalyzes the formation of an intermolecular isopeptide bond between Gln137 and Lys122.4 Upon this intermolecular cross-linkage, the RP S19 oligomers gain the extra-ribosomal function of monocyte/ macrophage-selective chemoattraction.5 This results in the generation of monocyte chemotactic capacity in serum in vitro.2,6,7 RP S19 oligomers, but not monomers, exhibit monocyte chemoattraction by acting as a ligand for the C5a receptor.5 Monocyte/macrophage-selective recruitment is provided by the dual effects of RP S19 oligomers as agonists of the C5a receptor of monocytes/macrophages and antagonists of the C5a receptor of neutrophils.5 The neutrophil-selective antagonist effect is attributed to the C-terminal 12 amino acid residues of RP S19,8,9 and a recombinant chimeric protein C5a/RP S19, in which the RP S19 C-terminal 12 residues are connected to the C-terminal of C5a, reproduces the dual effects of the RP S19 oligomers.10 We previously developed the coagulum absorption model in the peritoneal cavity of guinea pig to examine the biological role of the RP S19 oligomers generated in the blood coagulum. After intra-peritoneal transplantation, the coagulum is covered and infiltrated by macrophages within a day, and coagulum components are engulfed by the infiltrated macrophages.11 Currently, we prepared a homozygous gene knock-in mouse in which the RP S19 gene was replaced by a Gln137Glu-RP S19 artificial gene. The Gln137Glu mutation seems to cause dysfunction specific to the extra-ribosomal function of RP S19. In the current study, we have reexamined the role of RP S19 in blood plasma using the knock-in mouse-derived materials.
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Importance of macrophages in biological events was sometimes shown by the macrophage-depletion experiment. A technique to deplete macrophages from mice using liposome encapsulating clodronate (dichloromethylenebisphosphonate) which is an ATP mimetic reagent has been established,12 and the clodronate liposome is now commercially available. In the current study, we used clodronate liposome-treated mice to demonstrate the importance of macrophages in the intraperitoneal coagulum resorption model.
C57BL/6J and intercrossed the heterozygous Gln137GluRP S19 knock-in mice to obtain homozygous mice. We confirmed homozygous Gln137Glu-RP S19 knock-in by PCRSouthern blotting analysis. The homozygous knock-in mouse was fertile and apparently grew normally. The wild type and knock-in mice were maintained in the Center for Animal Resources and Development, Kumamoto University. The animal experiments were performed under the control of the Ethical Committee for Animal Experiment, Kumamoto University School of Medicine (approval number: B23-165).
Monocyte chemotaxis assay
MATERIALS AND METHODS Proteins and reagents We obtained rabbit IgG conjugated with fluorescein isothiocyanate (FITC) from Sigma-Aldrich (St. Louis, MO). We prepared recombinant C5a and recombinant chimeric protein C5a/RP S19 as described previously.10 We purchased all other chemicals from Nacalai Tesque (Kyoto, Japan) or from Wako Pure Chemicals (Osaka, Japan) unless otherwise specified.
Preparation of Gln137Glu-RP S19 knock-in homozygous mouse bearing C57BL/6J background We prepared Gln137Glu-RP S19 knock-in homozygous mice bearing a C57BL/6J genetic background. We purchased specific pathogen-free Crij:CD1 (ICR) and C57BL/6J mice (15– 20 g body weight range) from Charles River (Yokohama, Japan). We utilized a BAC#24 158H15 clone (BACPAC Resources Children’s Hospital Oakland, Oakland, CA, USA) bearing mouse RP S19 exon 5, which codes for Gln137. Using this clone we prepared Gln(CAG)137Glu(GAG)mutated exon 5 by means of polymerase chain reaction (PCR), and the PCR product was inserted into pGEM-T Easy vector (Promega, Tokyo, Japan). We replaced wild type exon 5 in a RP S19 gene fragment in pBS vector by the mutant exon 5 with a marker cassette (Lox71-SA-IRES-neomysinpA-Lox71).13 After amplification of the mutant exon 5 in host DH5α cells, we cut off the mutated RP S19 gene fragment with restriction enzyme SacI. We homologously replaced the mutated RP S19 gene fragment to the same position as the wild type RP S19 gene of embryonic stem cells of TT2KTPU8 F1 mice using electroporation. We implanted the mutant RP S19 embryonic stem cells along with germinal cells of ICR mice into endometrium of female mice and mated the ICR mice progeny exhibiting 100% chimerism with Cre C57BL/6J transgenic mice to remove the marker cassette.13 We repeatedly backcrossed the knock-in heterozygous mice with wild type mice to adjust the genetic background to
We obtained peripheral blood into a heparinized syringe from healthy volunteers after getting their informed consent under approval of Institutional Review Board of Kumamoto University (approval number 673). We isolated mononuclear cells from the heparinized blood using Ficoll-Paque Plus (Amersham Biosciences KK, Tokyo, Japan). The mononuclear cell fraction contained approximately 20% monocytes when identified as the macrophage specific esterase positive cells.9 We evaluated the monocyte-attracting capacity of mouse sera using a 48-well chemotaxis chamber with a polycarbonate filter (5 μm pore size) (Neuro Probe, Inc., Gaithersburg, MD, USA) according to the method of Folk et al.14 After incubation for 90 min, we stained the membrane with Giemsa solution and counted the total number of monocytes that migrated beyond the lower surface of the membrane in five high-power microscopic fields. We have previously described these methods in detail.15
Preparation of mouse blood and serum We obtained mouse blood from retro-orbital plexus of wild type C57BL/6J mice or the knock-in mice with 27 gauge needles and used to prepare coagulum and serum. To prepare serum, we allowed blood to spontaneously coagulate in sterilized glass tubes for 60 min at 22°C, followed by centrifugation at 4000 × g for 20 min at 22°C.
Intraperitoneal coagulum resorption model To prepare mouse blood coagulum, we initially mixed 10 μL of FITC-conjugated rabbit IgG in phosphate-buffered saline (PBS) as a marker (final concentration 2 mg/mL, SigmaAldrich, St. Louis, MO, USA), C5a/RP S19 in PBS (final concentration 10−8 M) or PBS alone with 100 μL aliquots of liquid blood. We then poured the mixture into a glass cylinder that was covered on one end with a sterilized parafilm sheet to form the coagulum. We performed a small midline lapa-
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rotomy on C57BL/6J mice (wild type and the knock-in) under ether anesthesia, inserted two coagula, sutured the abdominal incision, and provided water and food ad libitum for 3 days prior to performing a surgical operation to recover the coagulum. After recovery, coagula were weighed and fixed in 10% formalin. We have previously reported this method using guinea pigs in detail.11
Preparation of clodronate liposome-treated mice We attempted to deplete macrophages according to the method of van Rooijen and Sanders.12 We used liposome encapsulating clodronate and control liposome produced by Hygieia Bioscience (Minoo, Japan). We diluted an aliquot of each liposome (10 mg/mL) 10 fold with PBS upon the treatment of mice. We intraperitoneally injected with 200 μL of one of the working liposome suspensions into each mouse (10 mg liposome/kg body weight) once a day for 3 continuous days. Next day, we subjected the mice to the intraperitoneal coagulum insertion experiment. Because we often observed paralysis of hind extremities in clodronate liposome-treated male mice, we only used female mice for the clodronate liposome treatment.
Histological examination After being weighed, the coagula recovered from the peritonea were immediately fixed in neutralized 10% formalin for preparation of paraffin sections. Paraffin sections with 4 μm thickness were stained with hematoxylin and eosin in the usual way. The histologic specimens were observed under a microscope, Provis AX (Olympus, Tokyo, Japan) with a digital camera, Penguin 600CL (Pixera, Los Gatos, CA, USA).
Figure 1 Absence of monocyte chemotactic capacity in serum of Gln137Glu-ribosomal protein S19 (RP S19) gene knock-in mouse. Chemotaxis chamber assay was performed for mouse sera prepared from retro-orbital plexus blood of a wild type mouse (WT-serum) and of a Gln137Glu-RP S19 knock-in mouse (KI-serum) using human peripheral blood monocytes. Upon the chemotaxis assays the samples were diluted 10 fold (10), 100 fold (102) or 1000 fold (103) with phosphate-buffered saline (PBS) and used (open columns). As the negative and positive controls, PBS and a recombinant human complement C5a (C5a) at 10−8 M were respectively used (closed columns). After a 90-min incubation of the micro-well chamber, the chemotaxis membrane was stained with Giemsa solution and the total number of monocytes migrated beyond the membrane was counted in five microscopic high-power fields. Each sample was evaluated in micro-wells, and the total number of migrated monocytes is shown as the mean ± standard deviation. Results with 10 fold diluted samples were analyzed with the paired Student’s t-test (P = 0.001). We repeated the same experiments using 3 pairs of wild type mice and the knock-in mice. The results were reproducible and data of a representative experiment are shown.
Statistical analysis
capacity. Serum prepared from peripheral blood of wild type mice possessed monocyte chemoattractant capacity, as observed in human and guinea pig sera.2,6,7,11 In contrast, serum prepared from Gln137Glu-RP S19 knock-in mice did not exhibit monocyte chemoattraction at all (Fig. 1).
We performed statistical analyses using unpaired or paired Student’s t-tests. We considered P ≤ 0.05 to be statistically significant (*P ≤ 0.05, **P ≤ 0.01).
Delay of resorption of Gln137Glu-RP S19 knock-in mouse-derived coagulum in peritoneum
RESULTS Lack of monocyte chemotactic capacity in Gln137Glu-RP S19 knock-in mouse-derived serum During blood coagulation, RP S19 monomers in plasma are cross-linked by the factor XIIIa-catalyzed reaction, becoming homo-oligomers and gaining monocyte chemoattraction
We next performed the coagulum resorption experiment in the mouse peritoneal cavity. To control the effects of variability among animals, we inserted a pair of coagula intraperitoneally into wild type and knock-in mice. For an inserted pair, one coagulum was prepared from wild type mouse blood, and the other was from knock-in mouse blood. During the preparation of the knock-in mouse coagulum, we added FITC-labeled IgG to distinguish the two in the peritoneum after recovery. In the initial experiment, we mixed FITC-
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Figure 2 Delay of resorption of Gln137Glu-RP S19 knock-in mousederived coagulum in peritoneum. One coagulum was prepared with 100 μL of peripheral blood of a wild type mouse which had been mixed with 10 μL of fluorescent isothiocyanate (FITC)-conjugated rabbit IgG as a marker in PBS (2 mg/mL at the final), the other one was from the knock-in mouse blood pre-mixed with 10 μL of PBS. The two coagula were inserted as a pair into the peritoneal cavity (a) of a wild type mouse (n = 9) or (b) of a knock-in mouse (n = 6). Three days later, the coagula were recovered, checked for the fluorescent activity, and weighed. The closed circles indicate the weight of coagula. Each two circles connected by a bar were recovered coagula pair from an individual mouse peritoneum. There are significant differences between these groups when analyzed by the paired Student’s t-test (a, P = 0.005; b, P = 0.043). To neglect unknown adverse effect of FITCconjugated IgG, we repeated the experiment of (a) but premixing FITC-IgG into the knock-in mouse coagulum. (c) The closed column and the open column indicate weights of recovered knock-in micederived coagula and wild type micederived coagula, respectively (n = 5, P = 0.01 in paired Student’s t-test). (d) Macroscopic pictures of a representative pair recovered from a wild type mouse peritoneum are shown.
conjugated IgG into the wild type mouse-derived blood. Three days after insertion, we recovered and compared the weights of the two coagula. Regardless of the genotype of the host mouse, the knock-in mouse-derived coagulum was larger than the wild type (P = 0.005 in the cases of coagula from wild type mouse peritoneum and P = 0.043 in the cases of coagula from knock-in mouse peritoneum) (Fig. 2a,b). To neglect unknown adverse effect of FITC-conjugated IgG, we repeated the experiment using peritonea of wild type mice but mixing FITC-conjugated IgG into the knock-in mousederived blood. The results were basically the same. The knock-in mouse-derived coagulum was recovered with a significantly larger size than the wild type mouse-derived coagulum again (Fig. 2c, P = 0.01). Macroscopic images of a recovered coagula set are shown in Fig. 2d.
Promotion of Gln137Glu-RP S19 knock-in mouse-derived coagulum resorption by supplementation with C5a/RP S19 To confirm that the delay of absorption of the knock-in mouse-derived coagula was due to the absence of the RP S19 oligomers, we supplemented the coagula with C5a/ RP S19. We prepared two types of knock-in mouse-derived coagula; one contained 10−8 M C5a/RP S19 and the other contained FITC-conjugated IgG. We then inserted these coagula as a pair into wild type mouse peritonea and recovered both coagula 3 days later. In all pairs, the C5a/RP S19supplemented coagulum became smaller than the control coagulum (P = 0.027) (Fig. 3a). To neglect unknown adverse effect of FITC-conjugated IgG again, we repeated the experi-
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Figure 3 Promotion of Gln137Glu-RP S19 knock-in mouse-derived coagulum resorption by supplementation with C5a/RP S19. A coagulum containing C5a/RP S19 (10−8 M at the final) which is a functional analogue of the RP S19 oligomers (KI Coagula + C5a/RP S19) and a coagulum containing FITC-IgG (KI Coagula) were inserted as a pair into the peritoneum of a wild type mouse. Three days later, the coagula were recovered, checked for the fluorescent activity, and weighed. The closed and open circles indicate the weight of coagula. Each two circles connected by a bar were recovered coagula pair from an individual mouse. (a) There is a significant difference between these groups when analyzed by the paired Student’s t-test (P = 0.027). To neglect unknown adverse effect of FITC-conjugated IgG, we repeated the experiment but premixing FITC-conjugated IgG into the C5a/RP S19-supplemented coagulum. (b) The closed column and the hatched column indicate weights of recovered knock-in mice-derived coagula with or without C5a/RP S19 supplementation, respectively (n = 5, P = 0.003 in paired Student’s t-test).
ment but premixing FITC-conjugated IgG into the C5a/ RP S19-supplemented coagulum. The results were basically the same. The C5a/RP S19-supplemented coagula recovered in significantly smaller sizes (P = 0.003) (Fig. 3b).
Histological observation of coagula recovered from peritonea We examined the recovered coagula histologically. In the case of wild type mice-derived coagula, the surface was covered by macrophages, and the inside was infiltrated by macrophages when it was transplanted into both wild type and knock-in mouse peritonea. The surface macrophage layer then became multiple and finally changed to a granulation-like structure (Fig. 4a). Infiltrating macrophages would engulf red blood cells within the coagulum as previously observed in the guinea pig coagula using an electron microscope.11 This process was greatly delayed in the case of knock-in mouse-derived coagula, regardless of the genotype of the mouse used for transplantation (Fig. 4b,d). After supplementation with C5a/RP S19, the histologic picture of the knock-in mouse-derived coagula was similar to that of the wild type-derived coagula: the surface was totally covered by multiply-layered macrophages (Fig. 4c).
Influence of clodronate liposome pretreatment of mice to coagulum resorption To confirm the important role of peritoneal macrophages, regardless the resident or the exudate, in the coagulum resorption, we tried to deplete peritoneal macrophages and precursor cells of them by pretreatment of mice with clodronate liposome. We judged the macrophage depletion from histological change of the spleen such as significant red pulp atrophy concomitant loss of macrophages (data not shown). After treatment of mice either with clodronate liposome or with control plain liposome for 3 days, we intraperitoneally inserted a pair of knock-in mouse-derived coagulum and wild type mouse-derived coagulum, and recovered the coagula 3 days later in the same way as describe above. Weights of recovered coagula are shown in Fig. 5. In the control liposome-treated group, the weights of knock-in mice-derived coagula were still heavier than those of wild type mice-derived coagula, although the size difference between the two coagula types was smaller when compared to the result with non-pretreated mice (see Fig. 2a). Distinct from this, the sizes of knock-in micederived coagula were even smaller than those of wild type mice-derived coagula in the clodronate liposome pretreated group. Histologically, a poor macrophage infiltration was
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Figure 4 Histological observation of coagula recovered from peritonea. A piece of each coagulum was fixed in neutralized 10% formalin, and a paraffin section of it was stained with hematoxylin and eosin. A pair of coagula (a) from wild type mouse blood and (b) from the knock-in mouse recovered from wild type mouse peritoneum (see Fig. 2a). A pair of coagula prepared from knock-in mouse blood premixed (c) with C5a/RP S19 or (d) with 10 μL of FITC-conjugated IgG. In c and d images, difference of the surface covering macrophage layers is focused (see Fig. 3a).
evidently observed on the coagulum surface regardless the knock-in mouse-derived coagula or the wild type mousederived coagula. Interestingly enough, focal neutrophil infiltration was observed on the surface of knock-in mousederived coagula (Fig. 6).
DISCUSSION The presence of a ribosomal protein in circulating plasma possessing an anticipative extra-ribosomal function seems unique for RP S19.2,3,7,11 We prepared Gln137Glu-RP S19 knock-in mouse in which the extra-ribosomal function was diminished but the ribosomal function was maintained. Because the knock-in mouse was fertile and apparently grew normally, the extra-ribosomal function-specific diminishment
in the knock-in mouse is suspected. This was supported at least in part by the lack of the monocyte chemoattraction capacity in the knock-in mouse-derived serum (Fig. 1). The resorption of knock-in mouse-derived coagulum in the peritoneal cavity was delayed (Fig. 2) in concomitance with a poor macrophage infiltration (Fig. 4b,d). This difference is similar to the difference between plain guinea pig coagula and coagula containing neutralizing IgG against the RP S19 oligomers after 3 days in the guinea pig peritoneum.11 Meeting our expectation, the delayed resorption of knock-in mouse-derived coagulum was reversed by supplementation of C5a/RP S19, a substitute of the RP S19 oligomers,10 in the knock-in mouse-derived coagulum (Fig. 3) accompanied by enhanced macrophage recruitment (Fig. 4c). The present data indicate again the importance of the RP S19 oligomers as macrophage chemoattractant in the coagulum resorption.
© 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
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Figure 5 Influence of clodronate liposome pretreatment of mice to coagulum resorption. Wild type mice were intraperitoneally injected with 200 μL suspension of either liposome encapsulating clodronate (n = 6) (a) or control plain liposome (n = 4) (b) (10 mg liposome/kg body weight) once a day for 3 continuous days. Next day, a pair of knock-in mouse-derived coagulum and wild type mouse-derived coagulum was intraperitoneally inserted into each mouse. The marker FITC-conjugated IgG was mixed into the wild type micederived coagula in this experiment. Three days later, the coagula were recovered and weighed. The solid columns and open columns denote the weights of knock-in mice-derived coagula and the weights of wild type mice-derived coagula, respectively. Statistical analysis was performed using the paired Student’s t-test. ■, KI; □, WT.
The prompt resorption of the wild type mouse-derived coagulum was observed in the peritoneal cavity of the knock-in mouse as in the case of the wild type mouse. This suggests normal chemotactic function of macrophages in the knock-in
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mouse to the RP S19 oligomers. This has been separately confirmed in vitro (Zao et al., unpublished data). In the current study, we depleted peritoneal macrophages and their precursors by intraperitoneal injection of the clodronate liposome once a day for 3 continuous days. We had anticipated an equal delay of the resorption between the knock-in mouse-derived coagulum and the wild type-mouse derived one. However, the size of the recovered knock-in mouse-derived coagulum was even smaller (Fig. 5a). Histologically, the surface macrophage infiltration seemed equally poor between these coagula types. Focal neutrophil infiltration was observed on the surface of knock-in mouse-derived coagula in at least several sections (Fig. 6). Therefore, a possible mechanism which may explain the relatively faster resorption of the knock-in mouse-derived coagulum would be participation of neutrophils in the coagulum resorption under the absence of macrophages. The clodronate liposome treatment was reported not to affect the neutrophil number.12 In addition to this, the RP S19 oligomers antagonize the C5a receptor of neutrophils in the chemotactic response,5,8,9 and promote neutrophil apoptosis.16 These would support the less involvement of neutrophils in the RP S19 oligomer-contained wild type-derived coagulum. However, more experimental studies are required to confirm the involvement of neutrophils in the coagulum resorption especially under the absence of macrophages. The present study confirmed the importance of blood RP S19 in the macrophage-mediated resorption of coagulum. We believe that the Gln137Glu-RP S19 knock-in mouse would be a useful tool for examining the roles of blood RP S19 in the resolution of intravascular thrombotic diseases in the future.
Figure 6 Histological observation of coagula recovered from peritoneum of clodronate liposome pretreated mouse. Pairs of coagula recovered from peritonea of the clodronate liposome pretreated mice as described in Fig. 5 were fixed in 10% neutral formalin, and paraffin sections of them were stained with hematoxylin and eosin. A representative set of (a) wild type mouse-derived coagulum and (b) knock-in mouse-derived coagulum are shown. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
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ACKNOWLEGEMENTS We thank Ms T. Kubo for her technical assistance in the histological preparations. This work was supported by a Grant-in Aid for Scientific Research C (KAKENHI 24590484 to T. Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and by A-STEP (AS251Z01945Q to T.Y.) from Japan Science and Technology Agency. REFERENCES 1 Idol RA, Robledo S, Du HY et al. Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production. Blood Cell Mol Dis 2007; 39: 35–43. 2 Semba U, Chen J, Ota Y et al. A plasma protein indistinguishable from ribosomal protein S19: Conversion to a monocyte chemotactic factor by a factor XIIIa-catalyzed reaction on activated platelet membrane phosphatidylserine in association with blood coagulation. Am J Pathol 2010; 176: 1542–51. 3 Nishiura H, Tanase S, Tsujita K et al. Maintenance of ribosomal protein S19 in plasma by complex formation with prothrombin. Eur J Haematol 2011; 86: 436–41. 4 Nishiura H, Tanase S, Sibuya Y, Nishimura T, Yamamoto T. Determination of the cross-linked residues in homo-dimerization of S19 ribosomal protein concomitant with exhibition of monocyte chemotactic activity. Lab Invest 1999; 79: 915–23. 5 Nishiura H, Shibuya Y, Yamamoto T. S19 ribosomal protein cross-linked dimer causes monocyte predominant infiltration by means of molecular mimicry to complement C5a. Lab Invest 1998; 78: 1615–23. 6 Kukita I, Yamamoto T, Kawaguchi T, Kambara T. Fifth component of complement (C5)-derived high-molecular-weight macrophage chemotactic factor in normal guinea pig serum. Inflammation 1987; 11: 459–79.
7 Okamoto M, Yamamoto T, Matsubara S et al. Factor XIIIdependent generation of 5th complement component(C5)derived monocyte chemotactic factor coinciding with plasma clotting. Biochim Biophys Acta 1992; 1138: 53–61. 8 Shrestha A, Shiokawa M, Nishimura T et al. Switch moiety in agonist/antagonist dual effect of S19 ribosomal protein dimer on leukocyte chemotactic C5a receptor. Am J Pathol 2003; 162: 1381–88. 9 Nishiura H, Zhao R, Yamamoto T. The role of the ribosomal protein S19 C-terminus in altering the chemotaxis of leukocytes by causing functional differences in the C5a receptor response. J Biochem 2011; 150: 271–7. 10 Oda Y, Tokita K, Ota Y et al. Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively. J Biochem 2008; 144: 371–81. 11 Ota Y, Chen J, Shin M et al. The presence of ribosomal protein S19-like molecule in guinea pig plasma and its role in blood coagulum resorption. Exp Mol Pathol 2011; 90: 19– 29. 12 Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J Immunol Meth 1994; 174: 83–93. 13 Araki K, Araki M, Yamamura K. Targeted integration of DNA using mutant lox sites in embryonic cells. Nucleic Acids Res 1997; 25: 868–72. 14 Falk W, Goodwin RH Jr, Leonard EJ. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33: 239–47. 15 Matsubara S, Yamamoto T, Tsuruta T et al. Complement C4-derived monocyte-directed chemotaxis-inhibitory factor: A molecular mechanism to cause polymorphonuclear leukocytepredominant infiltration in rheumatoid arthritis synovial cavities. Am J Pathol 1991; 138: 1279–91. 16 Nishiura H, Zhao R, Chen J, Taniguchi K, Yamamoto T. Involvement of regional neutrophil apoptosis promotion by ribosomal protein S19 oligomers in resolution of experimental acute inflammation. Pathol Int 2013; 63: 581–90.
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