Rheumatoid Factor-Like Igm in Plasmodium Berghei (Apicomplexa: Haemosporida) Infections of BALB/C Mice
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FOLIA PARASITOLOGICA 50: 176–182, 2003 Rheumatoid factor-like IgM in Plasmodium berghei (Apicomplexa: Haemosporida) infections of BALB/c mice Reuel Robert Hook, Jr.1, Theodore J. Green1 and Melissa K. Stuart2 1Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA; 2Department of Microbiology/Immunology, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, Missouri 63501, USA Key words: Plasmodium berghei, malaria, rheumatoid factor Abstract. Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgG1, IgG2b, and IgG3 were not significantly different from those of control animals. Rheumatoid factor (RF) is an immunoglobulin that test their hypothesis, the researchers added exogenous reacts with the Fc region of IgG (Nordstrom et al. 1998). human RF-IgM to in vitro cultures of P. falciparum RF is found in patients with rheumatic conditions, most (Welch, 1897), and found that the antiglobulin facilitated notably rheumatoid arthritis, Sjögren’s syndrome, and the agglutination of merozoites coated with immune cryoglobulinaemia, diseases in which the prevalence rates human or Aotus monkey IgG. In a related study, Stuart and of elevated RF can exceed 90% (Lane and Gravel 2002). Green (1990) generated monoclonal IgM antibodies with In addition, RF is elevated in the serum of patients with RF-like activity from mice immunized with P. falciparum. non-rheumatic conditions, including those with persistent When added to parasite cultures, the IgM antiglobulins infections of bacterial, viral, and parasitic etiology (Lane enhanced the growth inhibitory effects of parasite-specific and Gravel 2002). In the latter group, IgM molecules with monoclonal IgG molecules bound to the parasite surface. RF-like activity (RF-IgM) may play a protective role in In some instances, the presence of monoclonal RF-IgM host immunity. In vitro and in vivo studies have shown correlated with an increase in the number of schizonts that that RF-IgM can enhance agglutination of IgG-coated failed to disperse merozoites. In other cases, parasitaemia parasites and viruses (Risemberg et al. 1969, Clarkson and remained low in the absence of the schizont inhibition Mellow 1981, Green and Packer 1984, Stuart and Green phenomenon, suggesting that RF-IgM contributes to host 1990), facilitate complement fixation (Carson 1984), cell protection not only by agglutinating merozoites, but enhance phagocytosis (Van Snick et al. 1978), and also by increasing the density of the antibody coat neutralise the infectivity of viruses and intracellular surrounding the parasites, thus interfering with parasite- parasites (Coutelier and Van Snick 1988, Stuart and Green erythrocyte interactions. 1990, Douvas et al. 1996). An in vivo model is needed to determine whether RF- Malaria is among the chronic diseases in which IgM can affect the outcome of malaria in an intact host. elevated serum levels of RF-IgM have been reported in While primate models are available (Houba and Allison humans (Houba and Allison 1966, Greenwood et al. 1966, Klein et al. 1971), costs associated with their use are 1971). In monkeys, secondary exposure to Plasmodium prohibitive. In this study, we have examined the knowlesi Sinton et Mulligan, 1932 (Houba and Allison production of RF-IgM in BALB/c mice infected by 1966) or P. cynomolgi Mayer, 1907 (Klein et al. 1971) Plasmodium berghei Vincke et Lips, 1948. Our results leads to rapid induction of RF-IgM. This observation suggest that a murine model would be suitable for prompted Green and Packer (1984) to speculate that examining the role of RF-IgM in immunity to malaria in resistance to malaria might be enhanced by RF-IgM. To vivo. Address for correspondence: M.K. Stuart, Department of Microbiology/Immunology, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, Missouri 63501, USA. Phone: ++1 660 626 2474; Fax: ++1 660 626 2523; E-mail: [email protected] 176 Hook et al.: Rheumatoid factors in rodent malaria MATERIALS AND METHODS was diluted 1:10 in sterile PBS containing 5 units of preservative-free heparin/ml, washed 4 times by centrifugation Parasites. A strain of Plasmodium berghei obtained from the for 10 min at 2000 × g at 4°C, and resuspended at 1:10 in sterile Walter Reed Army Institute of Research was maintained as PBS. Leucocytes were removed from the suspension by passage frozen stocks at –85°C or by in vivo passage in female BALB/c through a 1 × 10 cm column of packed cellulose fiber (Sigma) mice. For in vivo passage, blood specimens collected by cardiac previously equilibrated with sterile PBS. After centrifugation at puncture from infected animals were diluted 1:100 in Ventrex 2000 × g for 10 min at 4°C, the erythrocytes were lysed by HL-1 cell culture medium (Ventrex Laboratories, Portland, ME), resuspending the cells 1:10 in 0.83% ammonium chloride and and infected erythrocytes were counted by microscopic incubating them for 15 min in a 37oC water bath. Parasites were observation on a haemocytometer. To initiate new infections, washed with sterile PBS by centrifugation at 2000 × g for 10 blood specimens were diluted to contain 500 infected cells/ml in min at 4°C until the wash fluid was clear. The parasites were Ventrex HL-1 medium containing 2% foetal calf serum. resuspended in 3 ml of sterile PBS, overlaid onto 5 ml of Parasites were passaged twice in vivo before they were used in Histopaque-1077 (Sigma), and centrifuged at 400 × g for 30 min experiments. at room temperature. The band of parasites at the Histopaque- Infection of mice. Specific pathogen-free female BALB/c PBS interface was collected, washed 3 times with PBS by mice, aged 6 to 8 weeks, were used in this study. In the centrifugation at 2000 × g for 10 min at 4°C, and resuspended in experimental group, 50 mice were each injected intravenously 2 ml of sterile PBS. Parasites were lysed by sonication and (i.v.) with 50 P. berghei-infected erythrocytes in 0.1 ml of stored at –85°C until used as antigen in the ELISA. Protein Ventrex HL-1 medium. Ten uninfected control mice were determinations were performed by the BCA method (Pierce injected i.v. with 0.1 ml of medium alone. Five infected mice Chemical Co., Rockford, IL) using bovine serum albumin as were sacrificed on days 3 through 12 post-infection (dpi), while standards. two control mice were sacrificed on days 3 through 7 after ELISA plates (Falcon ProBind, Fisher Scientific, St. Louis, injection with diluent. Thin smears prepared from blood MO) were sensitized by overnight incubation at 4°C with specimens collected from each animal immediately prior to parasite antigen diluted to 100 ng/100 µl/well in 0.1 M carbonate sacrifice were stained with modified Wright-Giemsa stain and buffer, pH 9.5. The plates were blocked for 1 h at room microscopically examined to determine parasitaemia levels. At temperature in PBS containing 0.02% Tween 20 and 0.25% sacrifice, blood specimens were collected from each animal by nonfat dry milk (blocking buffer). Serum specimens from cardiac puncture for determination of serum RF-IgM and infected mice or mice injected with diluent alone were diluted parasite-specific IgG levels as described below. After allowing 1:10 in blocking buffer and incubated at 100 µl/well overnight the blood to clot overnight at 4°C, the serum was collected and at 4oC. As a negative control, serum from uninjected, naive centrifuged at 3000 × g for 10 min at 4°C to remove remaining female BALB/c mice was prepared in like manner and applied debris. The serum was diluted 1:2 in 0.1 M PBS, pH 7.4, to the ELISA plates. Following incubation, the plates were containing 0.02% sodium azide as a preservative, and stored at washed 3 times with PBS containing 0.2% Tween 20 (wash 4°C until assayed. buffer), and then incubated for 3 h at room temperature with 100 Spleen cell cultures were established from infected and µl/well of affinity-purified, horseradish peroxidase-labeled, Fc- control mice by teasing dissected spleen tissue into Ventrex HL- specific goat anti-mouse IgG secondary antibody (Jackson 1 medium. After 2 washes in the medium at 400 × g, an aliquot ImmunoResearch Laboratories, West Grove, PA) diluted of cells was removed, diluted 1:10 in 3% glacial acetic acid, and 1:10,000 in blocking buffer. After 4 washes with wash buffer, the number of nucleated cells determined by microscopic 100 µl/well of 2,2’-azinobis [3-ethylbenzothiazoline-6-sulfonic examination. Nucleated spleen cells were resuspended at 107 acid] (ABTS) (Sigma) were added, and the plates were incubated cells/ml in Ventrex HL-1 medium containing 2% foetal calf for 15 min at room temperature. Optical densities (O.D.) were serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml determined at 405/490 nm in an automated plate reader (Bio-Tek streptomycin.