Alternative versus classical macrophage activation during experimental African trypanosomosis.

African trypanosomes are extracellular parasites causing sleeping sickness to human or nagana to livestock in sub-Saharan Africa. To gain insight into factors governing resistance/susceptibility to these parasites, the immune responses in mice infected with a Trypanosoma brucei phospholipase C null mutant (PLC(-/-)) or its wild type counterpart (WT) were compared. We found that the T. b. brucei mutant inducing a chronic infection triggers the production of type I cytokines during the early stage of infection, followed by the secretion of type II cytokines in the late/chronic phase of the disease. In contrast, WT-infected mice are killed within 5 weeks and remain locked in a type I cytokine response. The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e. classically activated macrophages (type I) versus alternatively activated macrophages (type II) that are antagonistically regulated. Therefore, the phenotype and accessory cell function of macrophages elicited during WT and PLC(-/-) T. b. brucei infections were addressed. Results indicate that classically activated macrophages develop in a type I cytokine environment in the early phase of both WT and PLC(-/-) trypanosome infections. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated alternative macrophages. In parallel, we found that mice susceptible to Trypanosoma congolense infection, showing an exponential parasite growth until they die, have a higher level of type II cytokines in the early stage of infection than resistant animals controlling the first peak of parasitaemia. The levels of type I cytokines were comparable in both T. congolense-resistant and -susceptible mice. On the basis of these results, we propose that survival to African trypanosome infection requires a type I cytokine environment and classical macrophage activation in the early stage of infection, enabling mice to control the first peak of parasitaemia. Thereafter, a switch to type II cytokine environment triggering alternative macrophage activation is required to enable progression of the disease into the chronic phase. The possible role of the sequential activation of alternative macrophages in the late/chronic stage of infection in the increased resistance of mice to PLC(-/-) T. b. brucei will be discussed.

Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomosis.

Resistance to Trypanosoma brucei brucei has been correlated with the ability of infected animals to produce interferon (IFN)-gamma and tumor necrosis factor (TNF) in an early phase of infection, followed by interleukin (IL)-4 and IL-10 in late and chronic stages of the disease. Contributions of IFN-gamma and IL-10 in the control of parasitemia and survival of mice infected with T. brucei brucei were investigated by using IFN-gamma(-/-) and IL-10(-/-) mice. Results suggest that IFN-gamma, mainly secreted by CD8(+) T cells, is essential for parasite control via macrophage activation, which results in TNF and nitric oxide secretions. IL-10, partially secreted by CD4(+) T cells, seems to be important for the survival of infected mice. Its absence resulted in the sustained secretion of inflammatory mediators, which indicated the role of IL-10 in maintaining the balance between pathogenic and protective immune responses during African trypanosomosis.

Alternative versus classical macrophage activation during experimental African trypanosomosis.

The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e., classically activated macrophages (caMphi, type I) versus alternatively activated macrophages (aaMphi, type II). Recently, we showed that although mice infected with phospholipase C-deficient (PLC-/-) Trypanosoma brucei brucei exhibit a clear shift from type I to the type II cytokine production, wild type (WT)-infected mice remain locked in a type I cytokine response. In the present study, phenotype and accessory cell function of macrophages elicited during WT and PLC-/- T. b. brucei infection were compared. Results indicate that caMphi develop in a type I cytokine environment in the early phase of WT and PLC-/- trypanosome infection, correlating with inhibition of T cell activation triggered by a mitogen, a superantigen, or an antigen. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated aaMphi correlating with impaired antigen- but not mitogen- or superantigen-induced T cell activation.

Trypanosoma brucei brucei infection impairs MHC class II antigen presentation capacity of macrophages.

During African trypanosomiasis, macrophages play a central role in T cell hyporesponsiveness to parasite-related and unrelated antigens. In this study, the ability of macrophages from Trypanosoma b. brucei-infected mice to present exogenous antigens to a major histocompatibility complex (MHC) class II-restricted CD4+ T cell hybridoma was analysed. We demonstrate that the antigen presentation capacity of macrophages from infected mice is markedly reduced as a result of a lower expression of [MHC class II-peptide] complexes on their plasma membrane. This defect did not result from a decreased antigen uptake/catabolism, a reduced MHC class II and intercellular adhesion molecule 1 expression on the surface of macrophages, a decreased affinity of MHC class II molecules for antigenic peptides, a competition between exogenous and parasite antigens, or the generation of inhibitory peptides. Our data indicate that the step resulting in coexpression of processed antigens and MHC class II molecules is affected in T. b. brucei-infected mice. Additionally, macrophages from infected mice secreted IL-10 that in turn contributes to the impairment of T cell activation.

Hemozoin is a key factor in the induction of malaria-associated immunosuppression.

Infection-associated immunoincompetence during malaria might result from macrophage dysfunction. In the present study, we investigated the role of macrophages as target for immunosuppression during infection, using the murine Plasmodium c. chabaudi model. Special attention has been paid to the analysis of processing/presentation of protein antigens and presentation of peptides, using cocultures of peritoneal exudate cells (PECs) from infected mice and antigen-specific T-cell hybridomas. The results obtained indicate a defective processing of protein antigens that becomes maximal at acute parasitemias. In addition, macrophages from acutely infected mice suppress the interleukin-2 production by the antigen-activated T-cell hybridomas. This effect was independent of prostaglandin and nitric oxide production by the macrophage. The possible role of parasite components in the impaired accessory cell function of PECs was investigated and hemozoin, the end-product of the hemoglobin catabolism by intraerythrocytic malaria parasites, was found to induce similar infection-associated deficiencies in vitro. Moreover, hemozoin, was shown to mimic the immunosuppressive effects induced in PECs during in-vivo infections with P. chabaudi. In conclusion, we propose that hemozoin is a key factor in the malaria-associated immunosuppression, affecting both the antigen processing and immunomodulatory functions of macrophages.

Identification of a coelomic mitogenic factor in Eisenia foetida earthworm.

Coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) exerts a mitogenic activity on murine splenocytes. Total coelomic fluid was subjected to size-exclusion chromatography and a semi-purified mitogenic fraction (fraction 5) was isolated and further characterized. Both coelomic fluid and the semi-purified fraction 5 block concanavalin A (ConA)-induced spleen cell proliferation but exert a synergistic effect on LPS-triggered spleen cell proliferation. Using a polyclonal antiserum neutralizing the mitogenic activity of the semi-purified fraction 5, a 60-kDa component was identified and named CMF (coelomic mitogenic factor). CMF was found to bind ConA which could account for its ability to inhibit ConA-induced spleen cell proliferation. CMF is present in the coelomic fluid as a trimer of a 20-kDa protein. N-terminal amino acid sequence of monomeric CMF reveals partial sequence homology with phospholipase A2 (PLA2). Moreover, CMF-enriched coelomic fluid fraction 5 exerts phospholipase activity comparable with that of bovine pancreatic PLA2. Our results suggest that coelomic fluid of E. foetida contains a ubiquitous PLA2-like enzyme which might be involved in immune reactions in earthworms such as anti-bacterial mechanisms.

Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein.

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated alpha-Poxn2 and alpha-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The alpha-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.

Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade.

Coelomic fluid of Eisenia foetida earthworms contains a 42-kDa protein named coelomic cytolytic factor 1 (CCF-1) that was described previously to be involved in cytolytic, opsonizing, and hemolytic properties of the coelomic fluid. Cloning and sequencing of CCF-1 reveal significant homology with the putative catalytic region of beta-1,3- and beta-1,3-1,4-glucanases. CCF-1 also displays homology with coagulation factor G from Limulus polyphemus and with Gram-negative bacteria-binding protein of Bombyx mori silkworm, two proteins involved in invertebrate defense mechanisms. We show that CCF-1 efficiently binds both beta-1,3-glucan and lipopolysaccharide. Moreover, CCF-1 participates in the activation of prophenoloxidase cascade via recognition of yeast and Gram-negative bacteria cell wall components. These results suggest that the 42-kDa CCF-1 protein of E. foetida coelomic fluid likely plays a role in the protection of earthworms against microbes.

Trypanosoma brucei infection elicits nitric oxide-dependent and nitric oxide-independent suppressive mechanisms.

During murine Trypanosoma brucei infection, macrophages contribute significantly to the inhibition of T cell responses. Although nitric oxide (NO) was shown to play a central role in macrophage-mediated splenic suppression, macrophage-mediated lymph node suppression occurred in an interferon-gamma (IFN-gamma)-dependent manner. In this study, using NO inhibitor NG-monomethyl-L-arginine and anti-IFN-gamma antibodies, the relative contribution of NO and IFN-gamma to the active inhibition of ex vivo concanavalin A-induced T cell proliferation taking place in the spleen and the lymph nodes of T. brucei-infected mice was investigated. NO contributes to the suppressive activity of spleen and lymph node cells only during early-stage infection. The existence of NO-independent suppressive pathway was further evidenced in IFN-gamma(-/-)-infected mice. Spleen cells from such animals do not produce NO but exert significant suppressive activity during the whole course of infection. In contrast in the lymph nodes, no suppressive activity is recorded at any moment of infection. Moreover, addition of exogenous IFN-gamma to cultures containing lymph node cells from IFN-gamma(-/-)-infected mice does not impair proliferation despite NO production in such cultures. Thus during late-stage infection, an IFN-gamma-independent suppressive mechanism is elicited in the spleen, whereas in the lymph nodes, IFN-gamma is required yet not sufficient to inhibit T cell proliferation.

Active antitumor immunotherapy, with or without B7-mediated costimulation, increases tumor progression in an immunogenic murine T cell lymphoma model.

BW-Sp3 is a BW-5147-derived T cell lymphoma with limited immunogenicity since, despite regression of the majority of subcutaneous tumors, an important fraction of the animals will die from metastases. In the present study, the BW-Sp3 cells were transfected with genes encoding B7-1 or B7-2, known to be involved in the induction of T cell responses. The resulting transfectants exhibited a reduced tumorigenicity and did not cause mortality in the syngeneic recipients. Furthermore, immunization with the B7-1 or B7-2 transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTL) that lysed both the transfectants and the wild-type BW-Sp3 cells. Since the B7 transfectants were completely rejected in syngeneic recipients and induced potent CTL recognizing the wild-type BW-Sp3 cells, these engineered cells were considered as candidates for immunotherapy. Vaccinations with the B7-1 or B7-2 transfectants could completely protect the animals from metastatic disease when subsequently challenged with wild-type BW-Sp3 cells. Furthermore, immunization with the B7 transfectants could prolong the survival time of mice that had been challenged intravenously with BW-Sp3 cells. Surprisingly, however, when these transfectants, as well as the wild-type BW-Sp3 cells, were used for vaccination of tumor-bearing animals, the presence of the subcutaneous BW-Sp3 tumors clearly interfered with the outcome of immunotherapy, resulting in increased malignancy, as reflected by a higher incidence of progressing tumors and a reduced survival rate. Possible implications for immunotherapy in humans are discussed.

In vitro simulation of immunosuppression caused by Trypanosoma brucei: active involvement of gamma interferon and tumor necrosis factor in the pathway of suppression.

Experimental infections of mice with the African trypanosome Trypanosoma brucei lead to a profound state of T-cell unresponsiveness in the lymph node cell (LNC) compartment. This suppression is mediated by macrophage-like cells which inhibit interleukin 2 (IL-2) secretion and down-regulate IL-2 receptor expression (M. Sileghem, A. Darji, R. Hamers, M. Van de Winkel, and P. De Baetselier, Eur. J. Immunol. 19:829-835, 1989). Similar suppressive cells can be generated in vitro by pulsing 2C11-12 macrophage hybridoma cells with opsonized T. brucei parasites (2C11-12P cells). Cocultures of 2C11-12P cells and LNCs secrete higher levels of gamma interferon (IFN-gamma), and the hyperproduction of IFN-gamma was found to be confined to CD8+ lymphoid cells. Elimination of CD8+ cells from cocultures of 2C11-12P cells and LNCs restores the T-cell proliferative response. Furthermore, addition of neutralizing anti-IFN-gamma antibodies to the cocultures reduces the level of suppression and concomitantly restores the level of IL-2 receptor expression. Hence, IFN-gamma plays a cardinal role in this in vitro model for T. brucei-elicited immunosuppression. Cocultures of LNCs and 2C11-12P cells in a two-chamber culture system further demonstrated that cell-cell contact is required for hyperproduction of IFN-gamma and, moreover, that IFN-gamma cooperates with a 2C11-12P-derived diffusible factor to exert its suppressive activity. Finally, tumor necrosis factor alpha (TNF-alpha produced by 2C11-12P cells was found to be implicated in the hyperproduction of IFN-gamma, since addition of neutralizing anti-TNF-alpha antibodies to cocultures reduced the level of suppression and concomitantly abrogated the hyperproduction of IFN-gamma. Collectively, our findings indicate that T. brucei-elicited suppressive 2C11-12 macrophage cells differentially influence T-cell subpopulations: (i) CD8+ cells are signaled via cell-cell contact to produce IFN-gamma, and TNF-alpha is implicated in this process, and (ii) locally produced IFN-gamma and macrophage-released factors act in concert to inhibit CD4+ and CD8+ T-cell proliferative responses.

Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli.

The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUB1 and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the pre-S2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses.

Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms.

Total coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) is capable of lysing different mammalian tumor cell lines. This cytolytic activity is different from tumor necrosis factor (TNF)-mediated lysis and is not due to proteolysis. Total coelomic fluid was subjected to ion-exchange chromatography separation and a fraction with prominent cytolytic activity was used to elicit monoclonal antibodies that were screened for their capacity to neutralize the cytolytic effect of total coelomic fluid. One of the prepared neutralizing IgG antibodies was used for the immunoaffinity purification of a cytolytic factor from total coelomic fluid. SDS-PAGE and Western blot analyses revealed a protein band with an apparent molecular weight of 42 kDa. This cytolytic protein (termed CCF-1 or coelomic cytolytic factor 1) can be adsorbed on the surface of opsonized particles and may be involved in opsonizing and hemolytic effects of coelomic fluid.

Inhibition of T-cell responsiveness during experimental infections with Trypanosoma brucei: active involvement of endogenous gamma interferon.

Lymph node cells (LNC) from mice infected with Trypanosoma brucei contain macrophage-like cells that inhibit interleukin-2 receptor (IL-2R) expression (M. Sileghem, A. Darji, R. Hamers, M. Van De Winkel, and P. De Baetselier, Eur. J. Immunol. 19:829-835, 1989). Evidence that gamma interferon (IFN-gamma) is actively involved in (i) the inhibition of IL-2R expression and (ii) the generation of suppressive cells during infections with T. brucei is presented. First, despite an impaired T-cell mitogenic response, LNC from infected mice are hyperresponsive for IFN-gamma production. Second, addition of neutralizing anti-IFN-gamma antibodies to cocultures of normal LNC and suppressive LNC populations reduces the level of suppression and restores the level of IL-2R expression. Third, administration of anti-IFN-gamma to T. brucei-infected animals increases the blastogenic response and reduces the suppressive activity of LNC.

Redistribution of a murine humoral immune response following removal of an immunodominant B cell epitope from a recombinant fusion protein.

Immunization of different mice strains with a recombinant fusion protein composed of the vector-encoded N-terminal leader peptide CroLac (containing lambda Cro and LacI fragments) and a part of the transmembrane protein of HIV-1 (gp41) led to a high anti-CroLac humoral immune response. A detailed analysis of this response revealed the presence of an immunodominant, linear B cell epitope localized near the C-terminus of the CroLac fragment. The immune response seemed to be biased towards this epitope since few or no monoclonal antibodies (mAb) could be generated against the remaining part of CroLac and the gp41 fragment. Upon removal of the immunodominant region from the fusion protein the immune response was redirected and spread over the previously non-immunogenic regions. Consequently, we report a model system in which an immunodominant B cell epitope biases the immune response away from less immunogenic epitopes on the same molecule.

Differential presentation of hepatitis B S-preS(2) particles and peptides by macrophages and B-cell like antigen-presenting cells.

Different cell types, including dendritic cells, macrophages and Ia+ B cells, have been described to present soluble antigen (Ag) to T-cell hybridomas. However, it is still not clear whether these different cell types can act as antigen-presenting cells (APC) for complex and insoluble Ag such as viral particles. Using yeast recombinant hepatitis B S-preS(2)-containing particles, T-cell hybridomas were generated and used as a tool to study processing and presentation of antigen. Different types of APC were compared in regard to their capacity to process and present the protein-lipid composed S-preS(2) particles and the thereof derived T-cell epitope containing peptides by different types of APC. While a S-preS(2)-derived T-cell epitope containing peptide, which does not require processing, could be presented both by macrophage and B-cell like APC, the presentation of S-preS(2) particles required the presence of macrophages. The fact that B-cell like APC and macrophages behave differently with regard to the presentation of S-preS(2) particles suggest that the uptake and/or processing of this type of Ag by B-cell like APC and macrophages is different.

A monoclonal antibody against peripheral benzodiazepine receptor activities the human neutrophil NADPH-oxidase.

A murine monoclonal antibody, mAb 8523, raised against whole human pro-monocytic U937 cells recognizes an 18 kDa antigen in human neutrophils (PMN), as determined by immunoprecipitation and by immunodetection on Western blots of SDS-PAGE of PMN membrane fractions. That is 18 kDa antigen corresponds to the phagocyte peripheral benzodiazepine receptor (PBZDR) is evidenced by its co-migration with the 18 kDa covalently labeled PBZDR, detected by autoradiography, and their co-modulation upon phorbol-myristate-acetate activation of PMN. Purified mAb 8523 (IgG2b) is able to dose-dependently and specifically stimulate both the basal and the FMLP-induced oxidative burst of intact human PMN, assessed by luminol-amplified chemiluminescence. This property of the first described monoclonal antibody against PBZDR supports the implication of this receptor in NADPH-oxidase activation and consequently in phagocyte-dependent host defense mechanisms.

TNF-alpha mediated selection of macrophage-resistant gene-regulatory tumor variants.

In vitro macrophage- or TNF-alpha-mediated selection procedures on 3LL tumor cells have led to the selection of 3LL variants manifesting a highly reduced sensitivity towards the cytotoxic effects of both TNF-alpha and tumoricidal macrophages, while retaining the parental sensitivity to the cytolytic activity of (i) H2O2, (ii) macrophage-ADCC reactions and (iii) NK cells. A correlation was observed between the TNF-alpha binding capacity of the 3LL cell lines and their susceptibility towards macrophage- and TNF-alpha-mediated cytotoxicity, indicating that macrophage and TNF-alpha sensitivity may partially be regulated at the TNF-alpha receptor level. Further, the selected 3LL variants are gene-regulatory variants rather than cellular mutants, as upregulation of the TNF-alpha receptor by interferon-gamma (IFN-gamma) or 5'-azacytidine treatment resulted in an increased vulnerability of the selected 3LL variants to the killing activity of macrophages and TNF-alpha. The resistance of the 3LL variants to macrophage- and TNF-alpha-mediated cytotoxicity in vitro was reflected by a higher tumorigenic and metastatic potential in vivo. Therefore, the generation of TNF-alpha- and macrophage-resistant variants through immunoselection may contribute to the basic mechanisms of tumor progression and metastasis.