Hiroko Sugaya

Defective B-1 Cell Development and Impaired Immunity against Angiostrongylus cantonensis in IL-5Rα-Deficient Mice

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Ablation of eosinophils with anti‐IL‐5 antibody enhances the survival of intracranial worms of Angiostrongylus cantonensis in the mouse

Effects of depressed eosinophilia on the development of Angiostrongylus cantonensis in the mouse were studied using monoclonal rat anti‐mouse‐interleukin‐5 antibody (anti‐IL‐5). The administration of anti‐IL‐5 strongly depressed peripheral, cerebrospinal fluid (CSF) and medullary eosinophilic responses in mice infected with A. cantonensis, when compared with groups treated with phosphate‐buffered saline solution (PBS) alone or isotype‐matched rat IgG. There was no significant difference in A. cantonensis antigen specific IgG and IgE antibody responses between rat IgG treated and anti‐IL‐5 treated mice. Intracranial worm recovery in anti‐IL‐5 treated mice was consistently high throughout the course of the study and some worms migrated from the brain to the lungs. By contrast, almost all the intracranial worms in the mouse groups treated with PBS alone or rat IgG died before day 32. These data clearly indicate that IL‐5 is essential for eosinophil responses in A. cantonensis infected mice and also that eosinophils serve as a potential effector cell in the killing of the intracranial worms in mice.

mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-α through a mitogen-activated protein kinase pathway in human colon cancer cells

MUC2 mucin is a secretory glycoprotein which is produced from the intestinal goblet cells and is a major component of the intestinal epithelial mucus. The biological function of MUC2 mucin is considered to be the protection of intestinal epithelial surface, whereas the regulatory mechanism of MUC2 mucin production in immune response is not completely understood. We have studied the effects of cytokines, IL‐4, IL‐13 and TNF‐α, on the regulation of MUC2 mRNA in the human colonic cancer cell lines, LS174T and HT29. The quantitative reverse transcription‐polymerase chain reaction showed that single addition of IL‐4, IL‐13 and TNF‐α to cell culture induced about two‐fold increase of MUC2 mRNA level in LS174T cells. Interleukin‐4 and IL‐13 activated phosphorylation of mitogen‐activated protein kinase in LS174T cells. A specific inhibitor of mitogen‐activated protein kinase pathway, U0126, totally inhibited the increase of MUC2 mRNA by IL‐4 or IL‐13 in those cells. Therefore, mitogen‐activated protein activation of kinase is required for the increase of MUC2 mRNA by IL‐4 or IL‐13 in LS174T cells. In contrast to LS174T cells, only TNF‐α increased MUC2 mRNA through a mitogen‐activated protein kinase pathway in HT29 cells that express low levels of MUC2 mRNA. These findings sustain a novel phenomenon that MUC2 mRNA expression is differently controlled by IL‐4, IL‐13, or TNF‐α in LS174T and HT29 cells, whereas the mitogen‐activated protein kinase pathway plays a role in the MUC2 mRNA expression induced by those cytokines in both cell lines.

T-cell-dependent eosinophilia in the cerebrospinal fluid of the mouse infected with Angiostrongylus cantonensis

Summary Eosinophilia of the cerebrospinai fluid (CSF) in permissive (rats) and non‐permissive (mice) hosts infected with Angiostrongylus cantonensis, and the possible mechanism of the eosinophilia were studied. In three strains of thymic mice (ICR, ddY and BALB/c), the infection provoked a marked CSF eosinophilia starting at around day 12, reaching a peak level at day 20 and maintaining significantly high levels until day 35, In contrast, in athymic nude mice of BALB/c strain the infection failed to evoke this eosinophilia, suggesting T‐ceil dependence of murine CSF eosinophilia. Humoral antibodies did not correlate with the induction of eosinophilia. A time‐course study of worm recovery in the mouse brains indicated a gradual but consistent reduction in worm burden in accordance with the rapid rise in CSF eosinophil levels. Bone marrow eosinophilia occurred in mice at day 5, which preceded CSF eosinophilia. Jirds. a permissive but less susceptible host, developed a CSF eosinophilia with a peak level at day 17, but which declined rapidly following the peak. Permissive rat hosts developed significant peripheral and bone marrow eosinophilia at day 35 but their CSF eosinophilia was markedly less prominent than that of mice and jirds. These data clearly indicate that there are distinct differences in the mechanism of eosinophilia and eosinophilia‐inducing factors between permissive and non‐permissive hosts.

Eosinophilia and intracranial worm recovery in interleukin-5 transgenic and interleukin-5 receptor α chain-knockout mice infected with Angiostrongylus cantonensis

Abstract We infected interleukin-5 (IL-5)-transgenic (IL-5-Tg) and IL-5 receptor α knockout (IL-5Rα−/−) mice with Angiostrongylus cantonensis to determine the possible roles of IL-5 and eosinophils in A. cantonensis infection in mice. IL-5-Tg mice demonstrated significantly higher eosinophilia in bone marrow, blood and cerebrospinal fluid (CSF), lower intracranial worm recovery and smaller female worms than naive C3H/HeN mice. Both IL-5-Tg and C3H/HeN mice evoked antigen-specific serum and CSF IgA antibody responses as early as days 5 and 7 postinfection, respectively. Prominent eosinophil infiltration was noted around intracranial worms in the subarachnoid spaces of the mouse brains; eosinophils adhering to the worm surface were degranulated. In contrast, IL-5Rα−/− mice yielded a higher worm recovery than wild-type or heterozygous mice at day 20 postinfection and failed to provoke CSF eosinophilia. These findings indicate that A. cantonensis infection in the mouse causes IL-5 production and subsequent CSF eosinophilia, the latter probably being involved in the killing of intracranial worms.

The role of eosinophils in Angiostrongylus cantonensis infection

Angiostrongylus cantonensis is the causative agent of human eosinophilic meningoencephalitis in the Pacific Islands and Southeast Asia. Prominent eosinophilia in the cerebrospinal fluid (CSF) of the patients has been used as one of the diagnostic criteria for the disease but the role(s) of the CSF eosinophils has remained to be elucidated. In this article, Kentaro Yoshimura, Hiroko Sugaya and Kazuto Ishido discuss the involvement of CSF eosinophils in the killing of intracranial worms and the damage of the central nervous system of the hosts, and consider why eosinophils in A. cantonensis infection play a more important role in nonpermissive hosts (including humans) than in the permissive rat host.

Cytokine responses in mice infected with Angiostrongylus cantonensis

Abstract For determination of the kinetics of cytokine production and its possible role in host resistance to Angiostrongylus cantonensis in the mouse, Th1 [interleukin 2 (IL-2) and interferon gamma (IFN-γ)] and Th2 (IL-5 and IL-4) cytokine production in the cerebrospinal fluid (CSF), sera, and culture supernatants of spleen cells (SC) or cervical lymph-node cells (CLNC) of infected BALB/c and C57BL/6 mice was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA). IL-5 and IL-4 were detected in CSF of both strains, with a peak response occurring at around days 12–15 and 20 postinfection (p.i.), respectively. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay also revealed prominent IL-5 and IL-4 mRNA expression in T-cells but not in eosinophils in CSF. SC and CLNC stimulated with A. cantonensis young adult-worm antigen released IL-5 in vitro at and after day 20 p.i. Contrarily, IFN-γ production in CSF and SC or CLNC culture supernatants was almost negligible before day 30 p.i. IL-5, IL-4, and IL-2 production in culture supernatants was rather prominent in resistant C57BL/6 mice as opposed to susceptible BALB/c mice as assessed by the magnitude of increase over preinfection levels. Antigen-specific IgG1 (but not IgG2a) responses were more prominent in C57BL/6 mice than in BALB/c mice. These data suggest that systemic and local Th2 cytokine responses, especially those involving IL-5, are predominant in A. cantonensis-infected mice and that IL-5 is an important cytokine underlying the innate resistance of the mouse against A. cantonensis.

Ultrastructural and morphometric analyses of eosinophils from the cerebrospinal fluid of the mouse and guinea‐pig infected with Angiostrongylus cantonensis

Summary Guinea‐pigs infected with Angiostrongylus cantonensis developed pleocytosis and eosinophilia in the cerebrospinal fluid (CSF) at day 12 post‐infection (p.i.). showing a peak response at day 20 p.i., followed by a gradual reduction. Ultrastructural observations on CSF eosinophils from infected mice and guinea‐pigs revealed various signs of eosinophil degranulation after day 14 p.i., suggesting the exocytosis of lysosomal material. Morphometric analysis indicated that CSF eosinophils after day 22 p.i. contained fewer granules as well as smaller granules than those at days 14‐20 p.i. These data suggest that CSF eosinophils release granule constituents into the outside of the cells and these secretion products could interact with the intracranial worms and are probably related to worm death. As degenerative atrophy or partial loss of Purkinje cells and the spongy vacuolation of the white matter were noted in the cerebellum of infected mice, it was suggested that CSF eosinophils could be a possible cause of neurological disorders in angiostrongyliasis cantonensis.

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis.

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO(4)) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15-36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15-34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x(L) mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.

Different susceptibility to the IL‐3 induced‐protective effects between Strongyloides ratti and Nippostrongylus brasiliensis in C57BL/6 mice

Repetitive administration of recombinant IL‐3 induced protection against Strongyloides ratti but not against Nippostrongylus brasiliensis in C57BL/6 mice. Numbers of S. ratti were negligible from day 4 to day 6 post‐infection in mice injected with IL‐3, whereas N. brasiliensis burdens were almost equal from day 4 to day 6 between mice injected with IL‐3 or with medium. Mice treated with IL‐3 and then concurrently infected with S. ratti and N. brasiliensis were protected from intestinal S. ratti but not from N. brasiliensis. The numbers of intestinal mucosal mast cells were increased by the repetitive IL‐3 treatment on one day after the final injection and was augmented by subsequent infection with both nematodes.