IgM in human immunity to Plasmodium falciparum malaria.

Most studies on human immunity to malaria have focused on the roles of immunoglobulin G (IgG), whereas the roles of IgM remain undefined. Analyzing multiple human cohorts to assess the dynamics of malaria-specific IgM during experimentally induced and naturally acquired malaria, we identified IgM activity against blood-stage parasites. We found that merozoite-specific IgM appears rapidly in Plasmodium falciparum infection and is prominent during malaria in children and adults with lifetime exposure, together with IgG. Unexpectedly, IgM persisted for extended periods of time; we found no difference in decay of merozoite-specific IgM over time compared to that of IgG. IgM blocked merozoite invasion of red blood cells in a complement-dependent manner. IgM was also associated with significantly reduced risk of clinical malaria in a longitudinal cohort of children. These findings suggest that merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity.

IL-6 promotes CD4+ T-cell and B-cell activation during Plasmodium infection.

Humoral immunity develops in the spleen during blood-stage Plasmodium infection. This elicits parasite-specific IgM and IgG, which control parasites and protect against malaria. Studies in mice have elucidated cells and molecules driving humoral immunity to Plasmodium, including CD4+ T cells, B cells, interleukin (IL)-21 and ICOS. IL-6, a cytokine readily detected in Plasmodium-infected mice and humans, is recognized in other systems as a driver of humoral immunity. Here, we examined the effect of infection-induced IL-6 on humoral immunity to Plasmodium. Using P. chabaudi chabaudi AS (PcAS) infection of wild-type and IL-6-/- mice, we found that IL-6 helped to control parasites during primary infection. IL-6 promoted early production of parasite-specific IgM but not IgG. Notably, splenic CD138+ plasmablast development was more dependent on IL-6 than germinal centre (GC) B-cell differentiation. IL-6 also promoted ICOS expression by CD4+ T cells, as well as their localization close to splenic B cells, but was not required for early Tfh-cell development. Finally, IL-6 promoted parasite control, IgM and IgG production, GC B-cell development and ICOS expression by Tfh cells in a second model, Py17XNL infection. IL-6 promotes CD4+ T-cell activation and B-cell responses during blood-stage Plasmodium infection, which encourages parasite-specific antibody production.

Lymphotoxin alpha and tumour necrosis factor are not required for control of parasite growth, but differentially regulate cytokine production during Plasmodium chabaudi chabaudi AS infection.

Tumour necrosis factor (TNF) plays important roles in the pathogenesis of severe malaria, as well as in the generation of immune responses against malaria parasites. However, far less is known about the role of the closely related TNF family member lymphotoxin alpha (LTalpha) during malaria. We have used mice deficient in either TNF or LTalpha, as well as chimeric mice generated using donor bone marrow from these animals, to study the roles of these cytokines following Plasmodium chabaudi chabaudi AS infection. TNF and LTalpha were not required for the resolution of P. chabaudi chabaudi AS blood-stage infection. However, LTalpha, but not TNF, was necessary for early IFNgamma production and the regulation of IFNgamma production later in infection. A similar delay to that found for IFNgamma production was also observed for TNF production in LTalpha-deficient mice, compared with control mice. These results identify divergent roles for TNF and LTalpha in the regulation of host immune responses during P. chabaudi chabaudi AS infection.

Experimental models of cerebral malaria.

Malaria remains a major global health problem and cerebral malaria is one of the most serious complications of this disease. Recent years have seen important advances in our understanding of the pathogenesis of cerebral malaria. Extensive analysis of tissues and blood taken from patients with cerebral malaria has been complimented by the use of animal models to identify specific components of pathogenic pathways. In particular, an important role for CD8+ T cells has been uncovered, as well divergent roles for members of the tumor necrosis factor (TNF) family of molecules, including TNF and lymphotoxin alpha. It has become apparent that there maybe more than one pathogenic pathway leading to cerebral malaria. The last few years have also seen the testing of vaccines designed to target malaria molecules that stimulate inflammatory responses and thereby prevent the development of cerebral malaria. In this review, we will discuss the above advancements, as well as other important findings in research into the pathogenesis of cerebral malaria. As our understanding of pathogenic responses to Plasmodium parasites gathers momentum, the chance of a breakthrough in the development of treatments and vaccines to prevent death from cerebral malaria have become more realistic.

Bromelain activates murine macrophages and natural killer cells in vitro.

The innate immune response is critical for effective immunity against most pathogens. In this study, we show that bromelain, a mixture of cysteine proteases, can enhance IFN-gamma-mediated nitric oxide and TNFalpha production by macrophages. Bromelain's effect was independent of endotoxin receptor activation and was not caused by direct modulation of IFN-gamma receptors. Instead, bromelain either enhanced or acted synergistically with IFN-gamma receptor-mediated signals. These effects were seen in both RAW 264.7, a macrophage cell line, and primary macrophage populations. Bromelain also increased IL-2- and IL-12-mediated IFN-gamma production by NK cells. These results indicate a potential role for bromelain in the activation of inflammatory responses in situations where they may be deficient, such as may occur in immunocompromised individuals.

Bromelain modulates T cell and B cell immune responses in vitro and in vivo.

The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells, but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

CD95 is required for the early control of parasite burden in the liver of Leishmania donovani-infected mice.

In this study we show an increased incidence of T cell apoptosis in the liver and spleen of mice infected with Leishmania donovani. T cells from L. donovani-infected mice were found to be increasingly susceptible to CD95-mediated apoptosis in vitro, compared to controls. To test if suboptimal T cell function resulting from CD95-mediated apoptosis contributes to sustained parasite burden in L. donovani parasitized mice, B6.gld mice (lacking functional CD95 ligand) were infected with L. donovani. Surprisingly, at four different time points no difference in levels of T cell apoptosis in the spleen and liver was found between these mice and controls following intravenous delivery of L. donovani amastigotes, indicating that the CD95 / CD95L interaction is not essential for T cell apoptosis in the L. donovani-infected liver and spleen. However, B6.gld mice were increasingly susceptible to L. donovani infection, associated with less efficient granuloma formation in the liver and uncontrolled parasite growth in the spleen. Late in infection (day 56 post-infection), B6.gld mice had higher numbers of IFN-gamma-producing CD4(+) T cells in the liver and spleen, indicating a role for CD95 signaling in the homeostasis of this subset of cytokine-producing T cells in L. donovani-parasitized mice. Adoptive transfer of CD4(+) and CD8(+) T cells into recombinase activating gene 1 knockout (RAG-1(- / -)) recipients, revealed that CD95L expressed on CD4(+) T cells contributes to early control of L. donovani infection in the liver via mechanisms that are independent of granuloma formation and induction of apoptosis. These results indicate important roles for CD95 and CD95L that are unrelated to regulation of apoptosis in the early control of L. donovani infection.

B cell-deficient mice are highly resistant to Leishmania donovani infection, but develop neutrophil-mediated tissue pathology.

Resolution of Leishmania infection is T cell-dependent, and B lymphocytes have been considered to play a minimal role in host defense. In this study, the contribution of B lymphocytes to the response against Leishmania donovani was investigated using genetically modified IgM transmembrane domain (muMT) mutant mice, which lack mature B lymphocytes. When compared with wild-type mice, muMT mice cleared parasites more rapidly from the liver, and infection failed to establish in the spleen. The rapid clearance of parasites in muMT mice was associated with accelerated and more extensive hepatic granuloma formation compared with wild-type mice. However, the liver of infected muMT mice also showed signs of destructive pathology, associated with the presence of increased numbers of neutrophils. The role of neutrophils in controlling parasite growth in the viscera was determined by depletion with the mAb RB6-8C5. This treatment led to a dramatic enhancement of parasite growth in both the liver and spleen of muMT and wild-type mice. As assessed by transfer of both normal and chronic-infection serum, Ig protects microMT mice from destructive hepatic pathology, but minimally alters their resistance compared with wild-type mice. However, adoptive transfer of CD4+ and CD8+ T cells into recombinase activating gene 1 (RAG1-/-) recipients, suggested that T cell function was not altered by maturation in a B cell-deficient environment. Taken together, these data suggest an inhibitory role for B lymphocytes in resistance to L. donovani unrelated to the presence or absence of Ig. However, Ig protects muMT mice from the exaggerated pathology that occurs during infection.

Enhanced hematopoietic activity accompanies parasite expansion in the spleen and bone marrow of mice infected with Leishmania donovani.

In this study, we have analyzed hematopoietic activity in the spleen, bone marrow, and blood of BALB/c and scid mice infected with Leishmania donovani. Our analysis demonstrates that infection induces a rapid but transient mobilization of progenitor cells into the circulation, associated with elevated levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) and MIP-1alpha. From 14 to 28 days postinfection, when parasite expansion begins in the spleen and bone marrow, both the frequency and cell cycle activity of hematopoietic progenitors, particulary CFU-granulocyte, monocyte, are dramatically increased in these organs. This is associated with increased accumulation of mRNA for GM-CSF, M-CSF, and G-CSF, but not interleukin-3. Our data also illustrate that hematopoietic activity, as assessed by changes in the frequency of progenitor cell populations and their levels of cell cycle activity, can be regulated in both a T-cell-independent and T-cell-dependent, as well as in an organ-specific, manner. Collectively, these data add to our knowledge of the long-term changes which occur in organs in which L. donovani is able to persist.

Organ-specific immune responses associated with infectious disease.

The immune response to infection can vary markedly in different organs of the same animal. In some organs, the infection can resolve with subsequent immunity to re-infection, whereas in other organs, pathogens can persist. Here, Christian Engwerda and Paul Kaye highlight the importance of defining organ-specific immune mechanisms for developing strategies that deal effectively with infectious diseases and their associated pathologies.

Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-alpha.

Alterations in hematopoiesis are common in experimental infectious disease. However, few studies have addressed the mechanisms underlying changes in hematopoietic function or assessed the direct impact of infectious agents on the cells that regulate these processes. In experimental visceral leishmaniasis, caused by infection with the protozoan parasite Leishmania donovani, parasites persist in the spleen and bone marrow, and their expansion in these sites is associated with increases in local hematopoietic activity. The results of this study show that L donovani targets bone marrow stromal macrophages in vivo and can infect and multiply in stromal cell lines of macrophage, but not other lineages in vitro. Infection of stromal macrophages increases their capacity to support myelopoiesis in vitro, an effect mediated mainly through the induction of granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha. These data are the first to directly demonstrate that intracellular parasitism of a stromal cell population may modify its capacity to regulate hematopoiesis during infectious disease. (Blood. 2000;95:1642-1651)

Bromelain, from pineapple stems, proteolytically blocks activation of extracellular regulated kinase-2 in T cells.

Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3epsilon mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-gamma, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.

Leishmania donovani infection initiates T cell-independent chemokine responses, which are subsequently amplified in a T cell-dependent manner.

Control of Leishmania donovani infection in immunocompetent mice is associated with hepatic inflammation and granuloma formation, both of which are absent in severe combined immunodeficient (scid) mice. In both BALB/c and scid mice, L. donovani infection induced a rapid hepatic accumulation of mRNA encoding macrophage inflammatory protein-1alpha (MIP-(1alpha), monocyte chemoattractant protein-1 (MCP-1) and interferon-gamma inducible protein-10 (gammaIP-10). This response was not preceded by increased IL-4 production in either strain, unlike that reported in other infectious disease models. Interestingly, only gammaIP-10 mRNA was maintained at elevated levels throughout the first 7 days of infection, by mechanisms involving CD4+ and CD8+ T cells, and CD4+CD8+ cells not activated in scid mice. By in vivo depletion and reconstitution of scid mice it was demonstrated that T cells regulate the expression of all three chemokines studied, while they themselves only produce gammaIP-10 in appreciable quantities.

Blockade of CTLA-4 enhances host resistance to the intracellular pathogen, Leishmania donovani.

CTLA-4 has recently been shown to act as a negative regulator of T cell activation. Here we provide evidence that blockade of CTLA-4 can result in enhanced host resistance to an intracellular pathogen. The administration of anti-CTLA-4 mAb 4F10 to BALB/c mice, 1 day following infection with Leishmania donovani, enhanced the frequency of IFN-gamma and IL-4 producing cells in both spleen and liver, and dramatically accelerated the development of a hepatic granulomatous response. The expression of mRNA for the CXC chemokine gammaIP-10 was also elevated above that seen in control Ab treated mice, and was directly correlated with the frequency of IFN-gamma producing cells. In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) mRNA levels were unaffected by anti-CTLA-4 treatment, suggesting that CTLA-4 blockade may exert selective effects on chemokine expression. These changes in tissue response and cytokine/chemokine production were accompanied by a 50 to 75% reduction of parasite load in the spleen and liver of anti-CTLA-4-treated animals compared to controls. Furthermore, administration of anti-CTLA-4 mAb 15 days after L. donovani infection, when parasite burden is increasing in both organs, also resulted in enhanced resistance. Thus, these studies indicate a potent immunomodulatory and potentially therapeutic role for interventions targeted at CTLA-4.

Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani.

IL-12 plays a key role in stimulating both innate and antigen-specific immune responses against a number of intracellular pathogens. A neutralizing anti-IL-12 monoclonal antibody (mAb) was used to define and compare the role of endogenous IL-12 in the liver and spleen of mice infected with Leishmania donovani. IL-12 neutralization both early and late in infection caused delayed resolution of parasite load, a transient decrease in IFN-gamma, IL-4, TNF-alpha and inducible nitric oxide synthase (NOS-2) production, and suppressed tissue granuloma formation in the liver of genetically susceptible BALB/c mice. In contrast to the liver of BALB/c mice, neutralization of IL-12 had no effect on parasite burden in the spleen over the first 28 days of infection. However, IL-12 appeared to be critical for the development of mechanisms which subsequently contain the growth of persistent parasites in this organ in that neutralization of IL-12 dramatically enhanced parasite growth after day 28 of infection. Following IL-12 neutralization, the later unchecked growth of parasites in the spleen was coincident with an extensive breakdown of the tissue microarchitecture. Immunohistochemical studies revealed that IL-12 was largely produced by uninfected cells in L. donovani-infected BALB/c mice. In contrast, the course of infection in the liver and spleen of genetically resistant CBA/n mice was unaffected by the administration of anti-IL-12 mAb. These results suggest that the liver and spleen in susceptible BALB/c mice have different temporal requirements for IL-12 in controlling L. donovani infection, whereas IL-12 plays little role in either organ in resistant CBA/n mice. In addition, IL-12 appears to be involved in the generation of both Th1 and Th2 responses during L. donovani infection in BALB/c mice.

Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection.

Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-gamma from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-alpha production is induced by day 3 post infection. Not only do these data satisfactorily explain the differences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.

B7-2 blockade enhances T cell responses to Leishmania donovani.

Infection with Leishmania donovani has been reported to induce a dominant Th1-type response in all strains of mice examined, providing a model for examining the regulation of Th1 responses in the relative absence of Th2 cross-regulation. Here we demonstrate that blockade of the costimulatory molecule B7-2, but not B7-1, has significant effects on disease progression, measured as day 28 parasite burden in the liver. The effects of B7-2 blockade were associated with increased IFN-gamma production, as determined 1) following restimulation with specific Ag, 2) by enumeration of IFN-gamma-secreting cells using ELISPOT assays, and 3) by analysis of IFN-gamma mRNA by reverse transcription-PCR. Surprisingly, IL-4-producing cells were also readily detected in infected mice by ELISPOT analysis. The frequency of these IL-4-producing cells and of IL-4 mRNA levels was also enhanced in the liver of infected mice treated with anti-B7-2 mAb. Administration of anti-B7-2 from the day of infection or delaying its administration until day 3 after infection had similar effects. Parasite-specific IgG1 and IgG2a responses were either unaffected or marginally increased following anti-B7-2 administration, contrary to the inhibitory effect of this treatment on responses to the T-dependent Ag DNP-BSA. These data support a model of T cell activation whereby B7-2/CD28 interactions play a relatively redundant role in initial T cell activation, but in which interference with later B7-2/CTLA4 interaction potentiates established cytokine responses.

Destruction of follicular dendritic cells during chronic visceral leishmaniasis.

Follicular dendritic cells (FDCs) play a pivotal role in the germinal center (GC) response and in the development and regulation of B lymphocytes. Pathologic changes in GCs and a loss of FDCs have previously been noted in various viral infections, notably HIV-1. However, such changes have not been formally described in a chronic parasitic infection. In BALB/c mice infected with Leishmania donovani, parasites persist in the spleen for long periods, with associated splenomegaly. To examine the fate of FDC during the course of this chronic infection, we used 1) immunohistology, with FDC-specific mAbs; and 2) passive immunization with immune complexes, followed by light and electron microscopy. This study demonstrates that destruction of FDCs and a concomitant loss of GCs are associated with chronic visceral leishmaniasis. These pathologic effects are notable from 4 wk postinfection. At 8 wk postinfection and beyond, FDC are almost undetectable by both immunohistology and functional immune complex trapping. The loss of FDCs is associated with the infiltration of heavily parasitized macrophages into the GC, and reduction in parasite burden by chemotherapy is able to retard the process of FDC destruction. These data directly demonstrate for the first time the loss of FDCs during a chronic parasite infection and suggest a mechanism underlying the aberrant regulation of B cell function in murine visceral leishmaniasis.