Parasite-specific immunoglobulin isotypes during lethal and non-lethal murine malaria infections

Production of parasite-specific antibodies is an important component of immunity to blood stage malaria infection, as shown by several previous studies in rodent models. However, no study has addressed the induction of humoral immunity by different parasites in a genetically homogeneous host population. Here, levels of parasite-specific immunoglobulin isotypes were measured during primary infections of Plasmodium chabaudi and of Plasmodium yoelii in inbred NIH mice inoculated with cloned lines of either avirulent or virulent erythrocytic parasites. Non-lethal infections were characterized by early and late significant upregulation of IgG2a and IgG1, respectively. In contrast, for lethal infections, a slower, reduced IgG2a response correlated with a rapidly fatal outcome prior to any significant synthesis of IgG1. It is proposed that the sequential upregulated synthesis of parasite-specific IgG2a (cytophilic) and IgG1 (non-cytophilic) is associated with protective immunity to blood stage malaria infections in mice. This may provide an immunological framework for examining humoral immunity to malaria in humans.     

Parasite-specific immunoglobulin isotypes during lethal and non-lethal murine malaria infections

Production of parasite-specific antibodies is an important component of immunity to blood stage malaria infection, as shown by several previous studies in rodent models. However, no study has addressed the induction of humoral immunity by different parasites in a genetically homogeneous host population. Here, levels of parasite-specific immunoglobulin isotypes were measured during primary infections of Plasmodium chabaudi and of Plasmodium yoelii in inbred NIH mice inoculated with cloned lines of either avirulent or virulent erythrocytic parasites. Non-lethal infections were characterized by early and late significant upregulation of IgG2a and IgG1, respectively. In contrast, for lethal infections, a slower, reduced IgG2a response correlated with a rapidly fatal outcome prior to any significant synthesis of IgG1. It is proposed that the sequential upregulated synthesis of parasite-specific IgG2a (cytophilic) and IgG1 (non-cytophilic) is associated with protective immunity to blood stage malaria infections in mice. This may provide an immunological framework for examining humoral immunity to malaria in humans.     

Macrophage cytotoxicity in lethal and non-lethal murine malaria and the effect of vaccination.

We investigated the development of cell-mediated immunity in lethal and non-lethal malarial infections by assaying the cytotoxic activity of spleen cells for L929 tumour cells at different times after infection of mice with the lethal P. berghei, a lethal variant of Plasmodium yoelii and the non-lethal P. yoelii and P. chabaudi. In all cases the cytotoxicity increased to a peak during the first week and then diminished but the time of the peak varied with the infection; its activity was lowest with P. berghei. A second peak occurred in the non-lethal infections at the time of recovery. A protective vaccine accelerated and enhanced the early peak of cytotoxicity. The activity was mediated by adherent phagocytic cells, probably through the release of tumour necrosis factor (TNF) by macrophages since it was inhibited by antiserum against recombinant mouse TNF and did not destroy TNF-resistant L929 cells. Its induction was not dependent on T cells since it occurred in T cell-deficient mice infected with non-lethal P. yoelii. However, the accelerated increase associated with vaccination could be adoptively transferred by spleen lymphocytes from vaccinated mice.     

Different chemokine expression in lethal and non-lethal murine West Nile virus infection

West Nile (WN) virus is a mosquito-borne flavivirus that can cause lethal encephalitis in humans and horses. The WN virus endemic in New York City (NY) in 1999 caused large-scale mortality of wild birds that was not evident in endemic areas in other parts of the world, and the pathogenesis of the WN virus strain isolated in NY (NY strain) appears to differ from that of previously isolated strains. However, the pathogenesis of NY strain infection remains unclear. This study examined CC (RANTES/CCL5, MIP-1 alpha/CCL3, MIP-1 beta/CCL4) and CXC (IP-10/CXCL10, B lymphocyte chemoattractant (BLC/CXCL13), and B cell- and monocyte-activating chemokine (BMAC/CXCL14)) chemokine expression during lethal NY strain and non-lethal Eg101 strain infection in mice. We found that the mRNA of the CC chemokines, RANTES, MIP-1 alpha, MIP-1 beta, and IP-10 was highly up-regulated in the brain of NY strain-infected mice. By contrast, BLC mRNA was not detected in either group of mice, and BMAC mRNA was highly up-regulated in late stage of infection with the non-lethal Eg101 strain relative to levels in NY strain-infected mice.     

Cytokine profile of murine malaria: stage-related production of inflammatory and anti-inflammatory cytokines

Balance between inflammatory and anti-inflammatory cytokines may be important in malaria presentation and outcome. To clarify cytokine interactions that produce pathology of malaria and control infection, C57BL/6 mice were infected with 10(4) parasitized RBCs from a non-lethal strain of Plasmodium yoelii. Kinetics was monitored showing the course of parasitemia, and cytokines were determined by RT-PCR from liver and spleen tissues. Inflammatory cytokines such as interferon-γ (IFNγ), interleukin (IL)-12, IL-6, tumor necrosis factor-α (TNFα) and anti-inflammatory cytokines, including IL-4 and IL-10, were investigated as key molecules that interact with immune cells in the activation of the immune responses. The production of IFNγ mRNA was found to be higher on day 7 than on day 21 after infection, and IL-12 and IL-6 showed higher expression in the liver than in the spleen. Though TNFα was highly expressed on day 14 after infection and on day 21 in the liver, such expression was decreased on day 21 in the spleen. Anti-inflammatory cytokines showed high expression in both the liver and spleen. The results suggest that a relative balance between inflammatory and anti-inflammatory cytokines is crucial and that the increase of inflammatory cytokine levels during the acute phase of malaria may reflect an early and effective immune response.The counteraction effect of anti-inflammatory cytokines is thought to play a role in limiting progression from uncomplicated malaria to severe life-threatening complications.     

Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria.

This study was undertaken to explore early differences in cytokine production during nonlethal and lethal blood-stage murine malaria infections. Cytokine analysis of spleens during these infections showed that the principal difference between two nonlethal and two lethal Plasmodium species was the production of gamma interferon 24 h after infection with nonlethal parasites. In contrast, no increases in interleukin-4 production were observed in the first 24 h and tumor necrosis factor alpha levels increased equally in both nonlethal and lethal infections. During the later phase of infection with nonlethal parasites, both gamma interferon and interleukin-4 levels increased markedly a few days before parasite clearance. Early increases in gamma interferon production in nonlethal infections of Plasmodium yoelii and Plasmodium chabaudi were dose related and increased significantly with the size of the inoculum. Studies with the nonlethal P. yoelii suggest that the early gamma interferon response is mediated by T cells and natural killer cells, as it was reduced in athymic mice and in mice depleted of their natural killer cells by treatment with specific antiserum. Infecting mice with increasing numbers of lethal P. yoelii and Plasmodium berghei parasites did not increase the amount of gamma interferon, interleukin-4, and tumor necrosis factor alpha produced in a dose-dependent fashion. We conclude that one consequence of the early production of gamma interferon and tumor necrosis factor-alpha, particularly after nonlethal P. yoelii infection, may be to adjust the balance of T-helper cell subset activation, and probably that of other immune responses, so as to enhance the mechanisms that are essential for elimination of the parasites. This suggests that a successful vaccine should contain antigens capable of inducing such responses.     

Differential involvement of non-specific suppressor T cells in two lethal murine malaria infections.

The suppression of the contact sensitivity of oxazolone in murine malaria is shown to be mediated by non-specific T suppressor cells, but to a different extent in infection caused by two different species of parasite. Depletion of T suppressor cells in vivo and/or anti-Thy 1.2 treatment in vitro indicated that in mice infected with P. berghei the suppressor effect was largely mediated by T cells. By contrast, in mice infected with a lethal strain of P. yoelii it was only partly due to T cells; B suppressor cells and/or macrophages may also be involved. However, depletion of T suppressor cells in vivo had no effect on the course of the parasitaemia or on the survival time. Therefore, we postulate that this kind of non-specific immunosuppression cannot be regarded as a major cause of lethality.     

Anti-lymphocyte antibodies in lethal mouse malaria. II. Induction of an autoantibody specific suppressor T cell by non-lethal P. yoelii

The anti-lymphocyte autoantibody response to irradiated lethal Plasmodium berghei malaria parasites in normal mice was significantly reduced when recipients were pre-treated with splenic T cells from mice recovered from a non-lethal Plasmodium yoelii infection. Suppression was specific for the autoantibody and did not affect the antibody response to the parasite. Experiments involving sequential P. yoelii-P. berghei infections in situ revealed that recovery from P. berghei was possible when the interval between the two infections was 14 days or more. This ability to recover from P. berghei correlated with a progressive reduction of anti-lymphocyte autoantibody suggesting a useful role for the suppressor cell. The possible link between suppressor cells and anti-lymphocyte autoantibodies in malaria is discussed.     

Cytokine production and apoptosis among T cells from patients under treatment for Plasmodium falciparum malaria

Available evidence suggests that Plasmodium falciparum malaria causes activation and reallocation of T cells, and that these in vivo primed cells re-emerge into the periphery following drug therapy. Here we have examined the cytokine production capacity and susceptibility to programmed cell death of peripheral T cells during and after the period of antimalarial treatment. A high proportion of peripheral CD3+ cells had an activated phenotype at and shortly after time of admission (day 0) and initiation of therapy. This activation peaked around day 2, and at this time-point peripheral T cells from the patients could be induced to produce cytokines at conditions of limited cytokine response in cells from healthy control donors. Activated CD8hi and TCR-gammadelta+ cells were the primary IFN-gamma producers, whereas CD4+ cells constituted an important source of TNF-alpha. The proportion of apoptotic T cells was elevated at admission and peaked 2 days later, while susceptibility to activation-induced cell death in vitro remained increased for at least 1 week after admission. Taken together, the data are consistent with the concept of malaria-induced reallocation of activated T cells to sites of inflammation, followed by their release back into the peripheral blood where they undergo apoptotic death to re-establish immunological homeostasis as inflammation subsides. However, the high proportion of pre-apoptotic cells from the time of admission suggests that apoptosis also contributes to the low frequency and number of T cells in the peripheral circulation during active disease.     

Early cytokine production is associated with protection from murine cerebral malaria

Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-gamma) at 24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-gamma, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN-gamma production at 3 to 4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN-gamma at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-gamma. Early IFN-gamma production was present in NK-cell-depleted, gammadelta-cell-depleted, and Jalpha281(-/-) (NKT-cell-deficient) mice but absent from beta2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN-gamma and CD8(+) T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.     

Cytokine production in the murine response to brucella infection or immunization with antigenic extracts.

In order to induce acquired cellular resistance (ACR) to facultative intracellular bacterial pathogens, infection with live organisms is required. It is possible that different cytokine responses to live bacteria or their extracted antigens could account for their different abilities to induce ACR. Therefore, mice were infected with live attenuated Brucella abortus vaccine strain 19, and their ability to produce cytokines, both in vivo and in vitro, was investigated over 12 weeks of infection. This was compared with the response to injection of soluble brucella proteins (SBP). During infection, serum levels of interleukin-6 (IL-6) were markedly increased over a period of 4 weeks during the peak of infection. SBP plus adjuvant induced a transient increase in serum IL-6. IL-1 and tumour necrosis factor-alpha (TNF-alpha) remained undetectable in both instances. Spleen cells taken at intervals after infection and cultured with brucella antigens produced high titres of IL-6, IL-1 and TNF-alpha. Immunization with SBP was less efficient than live infection at inducing these cytokines. Of the characteristically T-cell-derived lymphokines, interferon-gamma (IFN-gamma) production rose 2 weeks after infection, peaking at 6 weeks, while IL-2 was not detected until 6 weeks post-infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was produced in substantial amounts, but IL-3 production was minimal. In contrast, spleen cells from mice immunized with SBP produced IL-2 but failed to produce IFN-gamma. The implications of these results for the induction of ACR are discussed.     

Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.     

Cytokine production and dermatophytosis]

The characteristic pathological feature of dermatomycosis is numerous neutrophilic infiltrates within the epidermis. However, the precise mechanism of this infiltration remains unknown. In this study, interleukins 1 beta, 6, and 8, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF)-alpha levels in the medium where keratinocytes were co-cultured with Candida albicans, Malassezia and Trichophyton mentagrophytes, were determined by enzyme-linked immunosorbent assays (ELISAs) in order to estimate the effect of these fungi on the cytokine production from human keratinocytes. The IL-8 level in the supernatants increased with 1 to 14 hours of co-culture in response to live C. albicans, but the other cytokines were undetectable. Furthermore, the mRNA of IL- 8 in keratinocytes was also confirmed to increased. This data suggested that C. albicans directly induce interleukin 8 production from human keratinocytes without activated macrophages. The IL-6, IL-8, and TNF-alpha levels in the culture supernatants increased with 1 to 24 hours of co-culture with keratinocytes and Malassezia species but the MCP-1 level was undetectable. The IL-8 and TNF-alpha levels in the culture supernatants increased with 1 to 24 hours of co-culture with keratinocytes and Trichophyton mentagrophytes but the other cytokine levels were undetectable. The ELISA analysis of cytokine production by human keratinocytes will provide useful information in understanding the pathogenesis of dermatomycosis.     

T-independent macrophage changes in murine malaria

A study to investigate the participation of T cells in macrophage-mediated responses during malaria was performed in nude (nu/nu) and littermate (nu/+) mice infected with Plasmodium berghei (PB). We found that in both groups of mice spleen cells suppressed the mitogenic response to LPS. Both nu/+ and nu/nu infected mice also showed liver macrophage activation, reflected by increased plasminogen activator release. These findings suggest that at least some of the macrophage changes during malaria infection are T-independent.     

Specific lymphocyte transformation in murine malaria

Summary Lymphoblast transformation tests were performed on convalescent rats and mice, after infection with Plasmodium berghei had been reduced to latency. Spleen cells from immune animals reacted in vitro to specific plasmodial antigen and not to control antigen produced from non infected RBC. The response in vitro was dependent on the concentration of the antigen and the time of exposure to it. A correlation was observed between the parasitaemia of the convalescent animal during its acute infection and the reaction in vitro: the higher the parasitaemia the higher the in vitro stimulation.

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Zusammenfassung Lymphoblasten-Transformationsteste wurden an Ratten and Mäusen mit latenter Plasmodium berghei-Infektion ausgeführt.Die Milzzellen von diesen immunisierten Tieren reagierten in vitro mit spezifischem Plasmodium-Antigen, jedoch nicht mit einem Kontrollantigen, das in nicht infizierten Erythrocyten produziert wurde.Die Resultate dieses Tests in vitro waren von der Konzentration des Antigens und der Einwirkungszeit abhängig. Es wurde eine Korrelation der Parasitämie zwischen diesen Tieren während der akuten Infektion und der Reaktion in vitro festgestellt: Je höher die Parasitämie, desto höher die Stimulation in vitro.

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This work was done as part of the International Collaborative Research Programme on Malaria Immunology and Immunopathology supported by the Dutch government     

Blackwater fever like in murine malaria

Blackwater fever (BWF) is the term used to designate the occurrence of hemoglobin pigments in the urine of patients infected with malaria parasites. BWF is more often associated with Plasmodium falciparum infection in man. The pathogenesis of BWF has not been explained satisfactorily. In the present study, the clinical and pathological observations made upon CD1 mice infected with Plasmodium yoelii yoelii lethal strain with clinical signs of hemoglobinuria and acute renal failure were evaluated. From the 40 P. yoelii yoelii-infected mice, 14 presented hemoglobinuria. In the observations, it was emphasized that hemoglobinuria occurred in the animals 1–2 days before they die. At 6 days post-infection, infected hemoglobinuric mice (HM) exhibited clinical signs such as dark red urine, apnea, and evident oliguria and hematuria; urine microscopical examination showed very few red blood cells. The entire non hemoglobinuric infected mice had a high parasitemia preceding the time of death, while the HM parasitemia was just detectable. In HM, marked hepatosplenomegaly, anemia, and renal and hepatic dysfunction were observed with the blood chemistry analysis at 6 days post-infection. Severe renal lesions were demonstrated in histopathological and scanning electron microscopy samples. Occlusion and necrosis of convoluted tubules were the main lesions found. The conditions required for the experimental production of hemoglobinuria in CD1 mouse infected by P. yoelii yoelii is still unknown. The clinical picture of a BWF, like in our rodents, was produced exclusively by the interaction between the parasite and its host. Results showed that hemoglobinuria in CD1 mice infected with P. yoelii yoelii and BWF in man infected with P. falciparum are similar in their pathogenesis.     

Effect of recombinant human colony-stimulating factor on the course of parasitaemia in non-lethal rodent malaria

The effect of repeated subcutaneous injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the attenuatedPlasmodium berghei XAT infection in CBA mice was examined. When mice were injeected with rhG-CSF daily beginning 2 days before infection, the neutrophil count in the peripheral blood increased 5 times higher than that of control mice and the development of parasitaemia was suppressed significantly during the early phase of the infection. This suppressive effect of rhG-CSF was reduced by treatment of the mice with either anti-interferon (IFN)- or antitumour necrosis factor (TNF)- immunoglobulins. These results suggest that neutrophils may have a role in immunity against the parasites and that IFN- and TNF- are possibly involved.     

Cytokine production in whole bloodex vivo

Interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) have been reported to contribute to the pathogenesis of many inflammatory diseases, e.g., rheumatoid arthritis. As monocytes are believed to be the primary source of these cytokines in peripheral blood, the present study was conducted to establish ranges and patterns of IL-1 and TNF- secretion. Using heparinized unseparated whole blood obtained from normal human volunteers, peripheral blood monocytes were stimulated withSal. minnesota LPS or BSA/anti-BSA immune complex-coated beads (BSA-beads). ELISAs for IL-1 and TNF- were employed to quantitate cytokine levels in blood plasma without performing arduous and time-consuming extraction procedures. Over the course of a 6 hr incubation, LPS elicited a dose-dependent increase in TNF- and IL-1 production. Preincubation of whole blood with interferon- prior to the addition of a suboptimal dose of LPS or BSA-beads resulted in a synergistic potentiation of IL-1/TNF- production. Dexamethasone, utilized in the treatment of rheumatoid arthritis, proved to be a potent inhibitor of cytokine biosynthesis in whole bloodex vivo. The measurement of cytokine biosynthesis in a relevant physiologic environment not only avoids non-specific monocyte activation, but also may increase our ability to predict clinical outcomes in rheumatoid arthritis and/or other inflammatory diseases.