Complement-dependent B-cell activation by cobra venom factor and other mitogens?

It has been proposed that two distinct signals are required for the triggering of the precursors of antibody-forming bone marrow-derived cells (B cells): (a) the binding of antigen or of a mitogen to the corresponding receptor sites on B-cell membranes and (b) the interaction of activated C3 with the C3 receptor of B lymphocytes. There is growing evidence that B-cell mitogens and T (thymus-derived cell)-independent antigens are capable of activating the alternate pathway of the complement system (bypass). Therefore, the effect of another potent bypass inducer was investigated with regard to B-cell activation and the role of C3. Purified, pyrogen-free cobra venom factor was mitogenic for both T and B lymphocytes (cortisone-resistant mouse thymus cells and lymph node lymphocytes from congenitally athymic mice). Venom factor could substitute for T cells by restoring the potential of antibody formation to sheep red blood cells in mouse B-cell cultures supplemented with macrophages or 2-mercaptoethanol. Venom factor may be capable of conferring activated C3 to the C3 receptor of B lymphocytes: preincubation of lymphoid cells with homologous serum or plasma, 10 mM EDTA, and sepharose-coupled venom factor converted with serum to an enzyme active against C3, inhibited their capacity to subsequently form rosettes with sheep erythrocytes sensitized with amboceptor and C5-deficient mouse complement. In the absence of EDTA, preincubation of freshly prepared B-cell suspensions with C3-sufficient homologous serum also blocked their subsequent interaction with complement-sensitized erythrocytes and at the same time rendered them reactive to an otherwise T-cell-specific mitogen. Moreover, mitogen induced B-cell proliferation in lymph node (but not in spleen) cell cultures, appeared to depend on the availability of exogenous C3: zymosan-absorbed fetal bovine serum (only 8.3% site-forming units remaining) supported T-cell activation by phytohemagglutinin, concanavalin A, and venom factor, but failed to sustain B-cell stimulation by pokeweed mitogen, lipopolysaccharide, and venom factor. T-cell-dependent antibody formation in composite cultures containing T cells or T-cell-substituting B-cell mitogens, B cells, and macrophages, always required the presence of C3-sufficient serum.

Some aspects of IL-8 pathophysiology. III: Chemokine interaction with endothelial cells.

Chemokines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilized on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1 alpha on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.

Modulation of interferon-gamma-induced major histocompatibility (MHC) and CD14 antigen changes by lipophilic muramyltripeptide MTP-PE in human monocytes.

The lipophilic muramylpeptide derivative muramyltripeptide-phosphatidylethanolamine (MTP-PE, 0.05 to 5 micrograms/ml) and human recombinant interferon-gamma (rIFN-gamma, 1 to 100 U/ml) were applied singly or in combination to fresh human mononuclear blood leucocytes in vitro. After 15 to 72 hr incubation, culture- and drug-induced changes in beta 2-microglobulin (MHC class I associated), HLA-DR (MHC class II), and Leu-M3 (CD14) antigen expression were investigated by flow cytometry; changes in monocyte morphology (forward light scatter and side scatter) were assessed by scatter analysis. It was found that (1) rIFN-gamma caused a simultaneous down-regulation of the CD14 antigen and an up-regulation of MHC class I and class II molecules on the surface of cultured monocytes; (2) MTP-PE, which by itself failed to influence the expression of these antigens, synergized with rIFN-gamma in increasing MHC antigens and reducing CD14; (3) at high concentrations rIFN-gamma reduced monocyte viability to a small but significant extent and this effect was further potentiated by MTP-PE; and (4) untreated monocytes in culture showed an apparently MTP-PE-insensitive increase in size, density, and beta 2-microglobulin, HLA-DR, and CD14 antigen expression. The influence of MTP-PE on rIFN-gamma-induced surface marker changes may contribute to its immunoadjuvant activity in vivo.

Tissue localization of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes.

To determine the tissue localization of lymphocytes provisionally termed "complement-receptor lymphocytes," which are characterized by having a membrane receptor for antigen-antibody-complement complexes, we investigated the adherence of sensitized and nonsensitized sheep red cells to frozen sections of mouse lymphoid organs. Nonsensitized erythrocytes became bound exclusively to sinus-lining cells of spleen and lymph nodes, whereas erythrocytes sensitized with antibody and complement adhered to lymphocytes in the follicular areas and the marginal zone of the spleen and in the true cortex of lymph nodes. However, the doubly sensitized erythrocytes failed to bind to the "thymus-dependent" areas of peripheral lymphoid organs or to the thymus itself. We suggest that complement-receptor lymphocytes are of extrathymic origin and that they contribute substantially to follicular antigen localization, which appears to be complement-dependent.

Modulations of myelopoiesis in vivo by chemically pure preparations of cell wall components from gram-negative bacteria: effects at different stages.

Modulation of myelopoiesis by chemically pure preparations of different cell wall components from gram-negative bacteria was investigated in vivo. The effects of lipid A, outer membrane lipoprotein, and murein were evaluated at several distinct stages: induction of colony-stimulating activity (CSA) in the serum, increase in the number of committed splenic precursor cells (CFU-C) forming granulocyte-macrophage colonies in vitro, and triggering into the cell cycle of noncommitted hemopoietic stem cells (CFU-S) from bone marrow. The results reveal different patterns of activity of the bacterial cell wall components (BCWC) tested. (i) In C57Bl/6 mice and C3H/Bom mice, all three preparations were potent inducers of CSA. In C3H/HeJ mice, CSA was only induced by lipoprotein and murein and not by lipid A. After injection of lipid A or lipoprotein, but not murein, the number of CFU-C in spleens of C57Bl/6 mice was increased up to 100-fold. In C3H/Bom and C3H/HeJ mice, not only murein but also lipoprotein were much less potent in this respect. (iii) In C57Bl/6 mice, both lipid A and lipoprotein, but not murein, were capable of inducing the proliferation of CFU-S, as demonstrated by a hot thymidine cytocide technique. Thus, induction of CSA and changes in the pool size of splenic CFU-C after administration of BCWC may be unrelated events. On the other hand, the increase of CFU-C might reflect the mitogenicity of BCWC for CFU-S.

Trypsin increases in vitro antibody synthesis and substitutes for helper T cells.

Trypsin, a neutral protease, enhanced the direct plaque response of T cell-suffiecient mouse spleen cell cultures to sheep erythrocytes (SRBC) and significantly increased the number of spontaneous PFC against SRBC in cultures without antigen. Moreover, trypsin proved to be able to substitute for T cells in nu/nu spleen cell cultures stimulated with SRBC. Its restorative capacity in this type of response was comparable to the one of lipopolysaccharide. Restoration of antibody synthesis in T cell-deprived cultures could not be explained by enzymatic alteration of SRBC. The data are discussed in terms of a possible role of hydrolytic enzymes released by accessory cells during the induction of an antibody response.

Oral administration of muramyl dipeptide into mice modulates cell proliferation, immunoglobulin synthesis and cytokine mRNA levels in gut associated lymphoid tissues.

Muramyl peptides (MDPs) are synthetic immunostimulants capable of potentiating a multitude of immune functions following parenteral administration into a host. The parent molecule MDP was also found to exert certain activities when applied via the oral route. Thus, we have studied the effect of oral treatment of mice with MDP on the lymphoproliferative responses, immunoglobulin secretion and cytokine induction in gut-associated lymphoid tissues (GALT) employing lymphocyte transformation test, ELISA and RT-PCR, respectively. Cells derived from Peyer's patches (PP) of mice orally primed with MDP were found to have enhanced proliferative responses to different mitogens and to secrete significantly more IgG, IgM and IgA immunoglobulins than cells from unprimed mice. These effects were not observed with cells derived from mesenteric lymph nodes (MLN) or spleens of MDP-primed mice. However, oral administration of MDP resulted in the up-regulation of interleukin-6 (IL-6) mRNA in MLN and down-regulation of IL-4 and tumour necrosis factor-alpha (TNF-alpha) mRNAs in MLN, spleens and PP. These studies suggest that selective modulations of GALT responses by orally administered MDP are achievable and imply that these agents may be useful in the therapy and prophylaxis of allergic diseases.

Suppression of in vivo IgE and tissue IL-4 mRNA induction by SDZ 280.636, a synthetic muramyl dipeptide derivative.

Modulation of IgE isotype expression on B cells is one of the numerous effects of muramyl peptides on the regulation of the immune system. A non toxic diacyl glycerol derivative of muramyl dipeptide (MDP), in which the L-alanine is replaced by L-threonine (MDP-Threo-GDP; SDZ 280.636), is currently under investigation as lead compound for the development of an anti-allergic drug. In this report, the modulatory effect of orally administered SDZ 280.636 in a murine model on polyclonally induced IgE levels is described. In this model, mice are injected i.v. with goal anti mouse IgD (GAMD) and challenged three to four weeks later with goal IgG (GIG). Both the primary and secondary immune responses lead to an increase of serum IgE levels. We demonstrate the efficacy of this muramyl dipeptide derivative in selectively inhibiting a polyclonal IgE response in GAMD-primed, GIG challenged mice without affecting the levels of other immunoglobulin classes. It is further shown that the induction of interleukin 4 (IL-4) gene transcript levels in lymphoid organs, which is observed as a consequence of boosting GAMD pretreated mice with GIG, is selectively suppressed in gut associated lymphoid tissues (GALT) and mesenteric lymph nodes but not in spleen. In contrast, interleukin 13 (IL-13) mRNA levels are not affected by SDZ 280.636. The findings that SDZ 280.636 inhibits polyclonal IgE responses and suppresses IL-4, but not IL-13 mRNA expression point towards differences in the regulatory pathways of IL-4 and IL-13 gene transcription in lymphoid organs. Thus the mechanism of action appears to involve a specific suppression of IL-4 gene transcription in cells occurring in Peyer's patches and mesenteric lymph nodes which are among the first constituents of the immune system encountered by an orally administered drug.

Induction of tumouricidal leucocytes by the intranasal application of MTP-PE, a lipophilic muramyl peptide.

Single intranasal applications of MTP-PE, a lipophilic muramyl peptide, induce tumouricidal and tumouristatic leucocytes in the lungs of rats. In ex vivo assays the tumouristatic activity was detectable for 8 days after drug administration. By separation of the effector cells on Ficoll-Hypaque gradients, it was shown that both neutrophils and macrophages are responsible for this activity. Using the B16/BL6 melanoma system in mice, there was a high survival rate after repeated intranasal applications of MTP-PE.

Nor-MDP, saponin, corynebacteria, and pertussis organisms as immunological adjuvants in experimental malaria vaccination of macaques.

Vaccination of primates against malaria using antigen derived from erythrocytic parasite stages has been most successful where Freund's complete adjuvant has been employed. Since this adjuvant is clinically unacceptable its replacement is a matter of urgency.In the present work a muramyldipeptide derivative (nor-MDP) given in mineral oil has proved to be partially effective as an adjuvant for merozoite vaccination of Macaca mulatta against Plasmodium knowlesi, and saponin has proved to be effective in similar vaccination of M. fascicularis.

Selection of a muramyl peptide based on its lack of activation of nuclear factor-kappa B as a potential adjuvant for AIDS vaccines.

Activation of the cellular transcription factor nuclear factor-kappa B (NF-kappa B) by cytokines and other immunostimulants has been tightly linked with enhanced replication of human immunodeficiency virus-type 1 (HIV-1) in infected cells. Various immunomodulators are currently being examined in animal and human trials for their suitability as adjuvants in potential vaccines against acquired immunodeficiency syndrome (AIDS). It may prove to be beneficial to select adjuvants that do not induce NF-kappa B activation and particularly if the vaccines are to be aimed at seropositive individuals. We have examined a battery of synthetic immunostimulants of the muramyl peptide family for their ability to activate NF-kappa B in human and mouse cell lines. In this report, we demonstrate selective activation of NF-kappa B in different cell lines and by different muramyl peptides possessing immunostimulatory activities. The mechanism of such activation is apparently via production of reactive oxygen intermediates (ROI) since pretreatment of cells with antioxidants blocked subsequent activation of NF-kappa B. However, among all the molecules tested only one lipophilic, non-pyrogenic adjuvant active muramyl peptide showed a complete lack of NF-kappa B activation in all cell lines tested. This molecule could well become the adjuvant of choice in future AIDS vaccines.

Induction in mice of serum IgE levels after treatment with anti-mouse IgD antibodies is preceded by differential modulation of tissue cytokine gene transcription.

Injection of mice with purified goat anti-mouse IgD (GAMD) leads to an interleukin (IL)-4-dependent increase of serum IgE levels. Challenge of GAMD-primed mice with goat IgG (GIG) initiates a secondary immune response with elevated serum IgE. In this report, kinetic cytokine transcript profiles of murine lymphoid tissues in response to primary i.v. GAMD treatment, as well as GIG challenge are presented. For the first time, gene transcription patterns of the recently described cytokines IL-12 and IL-13 are shown and compared with the corresponding patterns for other cytokine genes involved in IgE regulation, i.e. IL-4, and interferon (IFN)-gamma. After GAMD injection, two groups of induction profiles were observed in spleen, mesenteric lymph nodes and Peyer's patches: while IL-4 and IL-12p35 gene transcription was strongly enhanced, IFN-gamma, IL-12p40 and IL-13 mRNA were only moderately induced. Generally, maximal mRNA induction was measured on days 3 to 4 after GAMD treatment. The data demonstrate a clear-cut difference between the IL-4 and IL-13 response on the transcriptional level although both gene products show similar biological activities. The cytokine mRNA profiles support the assumption of IL-4 playing the central role in generating an IgE response. However, they do not reflect a strict Th1 versus Th2 cytokine gene transcription pattern but rather point towards a concerted action of various, partially antagonizing cytokines with respect to the regulation of IgE synthesis. IL-12 may, possibly via stimulation of IFN-gamma synthesis, represent a counterbalancing factor in the IL-4-mediated IgE response.

Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide.

Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.

Kinetics and isotype profile of rheumatoid factor production during viral infection: organ distribution of antibody secreting cells.

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a- or IgG1-bound immunoglobulins isolated from the serum of virus-infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM-, IgG- and IgA-type IgG2a-reactive autoantibody secreting cells during the primary and memory anti-viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM-type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus-specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the viruspecific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a-specific IgM-type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti-viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype-switched autoantibodies.

Activation by some T-independent antigens and B cell mitogens of the alternative pathway of the complement system.

A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum. As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations. Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation). Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, [D-glutamic acid, D-lysin] copolymer, phytohemagglutinin and concanavalin A failed to activate C3. C3-consumption by concanavalin A was due to nonspecific binding.