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Macrophage type I and type II class A scavenger receptors (SR-A I/II) were initially identified and cloned as molecules that mediate the uptake of modified low-density lipoproteins.1–3 SR-A I/II can bind an extraordinarily wide range of ligands, including bacterial pathogens,4 and also function as cation-independent macrophage adhesion molecules.5,6 SR-A I/II has been shown to bind and facilitate bloodstream clearance of gram-negative bacterial endotoxin such as lipopoly-saccharide7 and lipoteichoic acid, an anionic polymer that expresses on the surface of most gram-positive bacteria.8 Studies with SR-A I/II knockout mice have revealed that SR-A I/II plays important roles in host defense against bacterial and viral infection, such as Listeria monocytogenes,6 Staphylococcus aureus,9 and Herpes simplex virus.6 Furthermore, SR-A I/II deficiency impair the uptake and elimination of Corynebacterium parvum by macrophages,10 and BCG (Bacillus Calmette Guerin)–infected mice lacking SR-A I/II are more susceptible to endotoxin shock in response to lipopolysaccharide.11 Thus, SR-A I/II plays a significant role in host defense against bacterial and viral infection. In protozoan infection, although Nogami and others12 have reported that SR-A I/II knockout mice with a ICR x 129 mixed genetic background were more susceptible than wild-type mice against Plasmodium berghei infection, little attention has been paid to strain differences for this receptor in terms of susceptibility to infection. In this study, we focused on the possible role of SR-A I/II against murine malarial infection, such as P. berghei and P. yoelli, by using SR-A I/II knockout mice with a C57BL/6J genetic background.
The genetic background of the SR-A I/II knockout mice6 was replaced by backcrossing into a C57BL/6J strain (CLEA Japan, Tokyo, Japan) for at least eight generations.13 Genotyping of the SR-A I/II mutation was examined by means of polymerase chain reaction (PCR). All reactions were carried out in MicroAmp reaction tubes with caps (N8010540; Applied Biosystems, Foster City, CA) in a final volume of 20 µL containing 2 µL of 10x PCR buffer, 2 µL of 2.5 mmol/L dNTP mixture, 0.4 µL of 0.05 mmol/L macrophage scavenger receptors (MSR)-sense primer (5'-TCAGGTGCAGAACACTT-CAGT-3'), 0.2 µL of 0.05 mmol/L macrophage scavenger receptors (MSR)-antisense primer (5'-TGCTTTGCTGTAGA-TTCACGG-3'), and 0.2 µL of 0.05 mmol/L Phospho Glycerin Kinase (PGK)-antisense primer (5'-GCTGTCCATCTG-CACGAGAC-3'). Taq DNA polymerase (20 U of EX Taq TaKaRa, Shiga, Japan) was used in each reaction. Thermocycling was performed in a Bio-Rad icycler. After an initial preheating step for 4 minutes at 94°C to achieve a hot start procedure,14 the first three cycles consisted of steps of 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 183 seconds. A touchdown procedure15,16 consisting of denaturation at 94°C for 30 seconds and annealing at 67–63°C for 30 seconds with a 1°C decrement per cycle was applied during the subsequent cycles (4–8). The 9–34 cycles each consisted of steps of 94°C for 30 seconds, 63°C for 30 seconds, and 72°C for 183 seconds. After the last cycle, an extension step of 72°C for 5 minutes was included. The amplified samples were subjected to agarose gel electrophoresis, resulting in detection of a 700-bp wild-type allele and a mutant allele with a size of 900 bp. SR-A I/II knockout and wild-type (C57BL/6J) mice were bred and housed in polycarbonate cages and maintained under a specific pathogen-free environment in light-controlled (lights-on, 0500–1900 hours) and air conditioned (temperature, 24 ± 1°C; humidity, 50 ± 10%) rooms. The mice had free access to standard laboratory chow (CA-1; CLEA Japan). Adult mice were used in this experiment.
The P. berghei NK65 and P. yoelii 17X strains were kindly provided by Dr. Waki (Gunma Prefectural College of Health Science) and Dr. Kobayashi (Kyorin University, School of Medicine), respectively. Parasites were frozen, stored, and subsequently maintained by a serial intraperitoneal inoculation of infected red blood cells (IRBCs) diluted with phosphate-buffered saline (PBS) in C57BL/6J mice every 3 days. Parasitized erythrocytes were collected from the blood of the infected mice, washed twice, and diluted with PBS at a concentration of 1 x 104 and 5 x 106 IRBCs/mL in P. berghei and P. yoelli, respectively. Both SR-A I/II knockout (N = 5–8) and C57BL/6J mice (N = 5–8) were inoculated with 0.2 mL of P. berghei or P. yoelli IRBC suspension by intraperitoneal injections. The course of infection was monitored daily or every other day to examine survival rates, parasitemia, hematocrit value, and the number of white blood cells (WBCs). Parasitemia was determined by calculating the percentage of erythrocytes infected with parasites on thin blood films stained with Giemsa. Hematocrit values and WBC counts were determined by using an auto hematology analyzer (Cell tac
, MEK-6358; NIHON KOHDEN, Tokyo, Japan). Infectivity experiments were replicated at least three times to confirm the reproducibility of results. The Animal Care and Use Committee of Obihiro University of Agriculture and Veterinary Medicine reviewed the protocols and confirmed that the animals used in this study were cared for and used under the Guiding Principals for the Care and Use of Research Animals promulgated by Obihiro University of Agriculture and Veterinary Medicine. Survival rates were analyzed statistically by Gehan generalized Wilcoxon method. Other data were analyzed by Student t test.
On day 9 after infection with P. berghei, 84% of SR-A I/II knockout mice and 28% of wild-type mice survived when they were inoculated with 2 x 103 IRBCs. However, all of the SR-A I/II knockout and wild-type mice infected with P. berghei died by 11 days after inoculation (Figure 1A
). There were no significant differences between the genotypes in survival rates after the P. berghei infection (P > 0.05). As shown in Figures 1B to D, similar kinetics in parasitemia, hematocrit value, and WBC counts were observed in both experimental groups. In P. yoelii infection, there were no significant differences between the genotypes in survival rates, parasitemia, hematocrit values, or WBC counts (Figures 2A to D).
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and 2). Furthermore, there were no differences between SR-A I/II knockout and wild-type mice in terms of infectious course after P. berghei NK65 and P. yoelli 17X infection (Figures 1 and 2). Taking together previous studies and findings presented here, the contribution of SR-A I/II function to host defense in malarial infection does not seem to be widely extended, and the effect of SR-A I/II might be exhibited in collaboration with other, as yet unknown, gene(s). However, SR-A I/II might be contribute to the host defense for a chronic infectious course in protozoan infection, as shown in the report of Nogami and others.12 Further studies will be required to examine the contribution of SR-A I/II in protozoan infection by using other mouse malaria such as P. berghei XAT and P. chabaudi.
Received August 31, 2005. Accepted for publication February 12, 2006.
Acknowledgments: The authors thank Y. Ueta, C. Ichikawa, M. Chiba, M. Kim, T. Ishijima, K. Maeda, T. Hori, D. Lee, J. Lee, and T. Asano for technical assistance for breeding animals. We are also grateful to Drs. Waki (Gunma Prefectural College of Health Science) and Suzuki (Gunma University, School of Medicine) for P. berghei NK65 and Dr. Kobayashi (Kyorin University, School of Medicine) for P. yoelii 17X. Pacific Edit reviewed the manuscript before submission.
Financial support: This study was supported, in part, by a grant from Special Coordination Fund for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science, Japan.
* Address correspondence to Hiroshi Suzuki, Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro, Hokkaido 080-8555, Japan. E-mail: hisuzuki{at}obihiro.ac.jp 
Authors’ addresses: Mai Inoue, Xuenan Xuan, Kozo Fujisaki, Ikuo Igarashi, and Hiroshi Suzuki, Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2–13, Inada, Obihiro, Hokkaido 080-8555, Japan. Hiroshi Suzuki, Department of Developmental and Medical Technology, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, E-mail: hisuzuki{at}obihiro.ac.jp.
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