Immunological properties of Fc receptor on lymphocytes. 5. Suppressive regulation of humoral immune response by Fc receptor bearing B lymphocytes.

High anti-DNP PFC responses to DNP-DE or DNP-KLH were obtained by transferring normal or primed FcR^? B cell fractions into irradiated syngeneic recipients. On the other hand, the FcR⁺ B cell fraction showed a low precursor activity. Trypsinization of the FcR⁺ B cells, to eliminate remaining antigen-antibody complexes on the surface, failed to augment the response in comparison with that of trypsin-untreated FcR⁺ B cells. Therefore, the weak precursor activity of FcR⁺ B cells seemed to be inherent. No synergistic interaction between the FcR⁺ B and precursor FcR^? B cells, to give rise to the maximum PFC response, was observed. On the contrary, the FcR⁺ B cells significantly suppressed the PFC responses of FcR^? B cells. This kind of suppression could be mediated by a factor released from the FcR⁺ B cell, but not from the FcR^? B or original-unrosetted spleen cell fraction. The factor was not attributable to macrophages, because the FcR⁺ B cells isolated from normal spleen cells, of which macrophages were depleted by Sephadex G-10 columns, could produce the factor with the same activity. Stimulation by specific antigen is not necessary for the induction of the factor(s) as well as of the suppressing FcR⁺ B cells. It seems to be necessary to stimulate FcR by antigen-antibody complexes to produce or release this factor.     

B cells

B cells are an important component of adaptive immunity. They produce and secrete millions of different antibody molecules, each of which recognizes a different (foreign) antigen. The fact that humans express a very large repertoire of antibodies is due to the complex mechanism of V(D)J recombination of immunoglobulin (Ig) genes as well as other processes including somatic hypermutation, gene conversion and class switching. The B cell receptor (BCR) is an integral membrane protein complex that is composed of two Ig heavy chains, two Ig light chains and two heterodimers of Igalpha and Igbeta. To eliminate foreign antigens, B cells cooperate with other cells of the immune system including macrophages, dendritic cells and T cells. B cell development is a tightly controlled process in which over 75% of the developing cells become apoptotic because of inappropriate immunoglobulin gene rearrangements or recognition of self antigens by Igs. Hence, the majority of B cell-associated disorders are caused by the incorrect function of genes/proteins involved in B cell development.     

Induction of the differentiation of memory T killer cells with factors released from macrophage-like cell lines

Macrophage-like cell lines J774.1 and WEHI-3 as well as peritoneal exudate macrophages have been demonstrated to produce factors which induce the differentiation of memory cells into specific T killer cells in the absence of an added antigen. LPS stimulation was required for J774.1 cells and peritoneal macrophages to produce the factors but not for WEHI-3 cells. Interferon seemed to be one of the responsible factors. However, macrophages seem to produce other active factors; one has a molecular weight (MW) of more than 80,000 and lacks thymocyte mitogenic activity; another, with a weak thymocyte mitogenic activity, has a MW of 38,000 to 44,000. The low MW thymocyte mitogenic factor (interleukin-1) showed weak T killer cell differentiating activity.     

Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.     

Macrophage factor controlling differentiation of B cells.

Peritoneal macrophages of the mouse produce, in response to cell wall components of Gram-negative bacteria (lipopolysaccharide and lipoproteins), a factor that causes antigen-stimulated B cells of differentiate into antibody-producing cells. Unlike lipopolysaccharide, this factor is not mitogenic for B cells. Production of the macrophage factor does not depend on participation of T cells or other accessory cells since it is readily produced by several cloned macrophage cell lines as well as by peritoneal macrophages of athymic nude mice. The factor is active only in conjunction with antigen. T cells, although apparently not necessary, amplify its effect. The factor induces phenotypic differentiation of B cell precursors as selectively as thymopoietin induces differentiation of prothymocytes.     

Changes in lymphocyte subsets and macrophage functions from high, short-term dietary ethanol in C57/BL6 mice

Chronic administration of a diet containing 7% ethanol (36% of total calories) for 8 days to male C57/BL6 mice resulted in significant changes in functioning of macrophages. Peritoneal exudate macrophages from the ethanol-fed mice released more tumor cell cytotoxic materials upon culturing in vitro than cells from controls. However, peritoneal exudate cells continued to respond to exogenous beta carotene in vitro to produce additional cytotoxic materials. Phagocytosis of sheep red blood cells in vitro was suppressed in cells from ethanol treated mice. The number of splenic lymphocytes of various subsets was significantly changed by the ethanol exposure. Total T cells and T suppressor cells were lower, with a significant decrease in B cells containing IgM on their surface. The percentage of spleen cells showing markers for macrophage functions and their activation were significantly reduced. It is concluded that short-term chronic consumption of dietary ethanol, which was sufficient to produce physical dependence, results in significant alterations in lymphocyte subtypes and suppression of some macrophage functions.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

The immune response in aged C57BL/6 mice. I. Assessment of lesions in the B-cell and T-cell compartments of aged mice utilizing the Fc fragment-mediated polyclonal antibody response

The Fc fragment-mediated polyclonal antibody response was utilized to assess B-cell, T-cell, and macrophage reactivity in aged C57BL/6 mice. Spleen cells from aged (28–30 months) mice were found to be deficient in their capacity to proliferate and produce polyclonal antibody in response to Fc fragments when compared to adult (2–3 months) controls. Since T cells are required for the Fc-induced polyclonal antibody response, T cells from aged mice were assessed for their ability to restore the polyclonal antibody response in T-cell-depleted adult spleen cell populations. Aged T cells were not as effective as adult T cells in restoring the antibody response. The T-cell requirement in the Fc-induced polyclonal response has been shown to be replaceable by the Fc-stimulated T-cell replacing factor (Fc)TRF. T cells derived from aged mice were unable to produce (Fc)TRF to the level of adult cells. In addition to a defect in the T-cell compartment a lesion exists in the B-cell compartment of aged mice as well. Adult T cells were not capable of restoring the polyclonal antibody response of aged B cells any higher than aged T cells indicating a B-cell defect. Moreover, when a direct B-cell activator, Fc subfragment, was employed, the aged B cells were not stimulated to the level of adult controls. To test the ability of aged macrophages to function as accessory cells in the polyclonal response, macrophage-depleted adult spleen cells were mixed with aged or adult macrophages and the response measured. The results indicate that aged macrophages restore the polyclonal antibody response as efficiently as their adult counterpart.     

Class II MHC antigen-positive macrophages from human placentae suppress strong MLR and CML reactions

Placental adherent cells (PAC), derived by mild enzymic digestion of human placentae and adherence to plastic, were tested for their ability to suppress MLR and CML reactions. Stimulation of allogeneic T cells by cord blood mononuclear cells or by a strongly stimulatory B lymphoblastoid cell line were consistently inhibited by all PAC preparations tested. PAC were fractionated by Fc rosetting and Percoll gradients to produce a number of bands. Suppression correlated with the content of macrophages in each band and was strongest in band 5 which generally contained 94-100% macrophages. The suppressive effect was not inhibited by indomethacin. The possible role of macrophages in suppressing immune reactivity in the placenta is discussed.     

Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: implications for Burkitt's lymphoma

Burkitt's lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.     

Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.     

Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss

The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha. In the present study, whether rainbow trout macrophages produce TNFalpha was examined. Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha. The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS. Results show that trout macrophage culture supernatants exhibit TNF-like activities. The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test. The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha. The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D. This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells. Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody. These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.     

Dietary fats affect macrophage-mediated cytotoxicity towards tumour cells

In the present study, the effects of feeding mice diets of different fatty acid compositions on the production of TNF-alpha and nitric oxide by lipopolysaccharide-stimulated peritoneal macrophages and on macrophage-mediated cytotoxicity towards L929 and P815 cells were investigated. C57Bl6 mice were fed on a low-fat (LF) diet or on high-fat diets (21% fat by weight), which included coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO) as the principal fat source. The fatty acid composition of the macrophages was markedly influenced by that of the diet fed. Lipopolysaccharide (LPS)-stimulated macrophages from FO-fed mice showed significantly lower production (up to 80%) of PGE2 than those from mice fed on each of the other diets. There was a significant positive linear correlation between the proportion of arachidonic acid in macrophage lipids and the ability of macrophages, to produce PGE2. Lipopolysaccharide-stimulated TNF-alpha production by macrophages decreased with increasing unsaturated fatty acid content of the diet (i.e. FO < SO < OO < CO < LF). Macrophages from FO-fed mice showed significantly lower production of TNF-alpha than those from mice fed on each of the other diets. Nitrite production was highest for LPS-stimulated macrophages from mice fed on the LF diet. Macrophages from FO-fed mice showed significantly higher production of nitrite than those from mice fed on the OO and SO diets. Compared with feeding the LF diet, feeding the CO, OO or SO diets significantly decreased macrophage- mediated killing of P815 cells (killed by nitric oxide). Fish oil feeding did not alter killing of P815 cells by macrophages, compared with feeding the LF diet; killing of P815 cells was greater after FO feeding than after feeding the other high fat diets. Compared with feeding the LF diet, feeding the OO or SO diets significantly decreased macrophage-mediated killing of L929 cells (killed by TNF). Coconut oil or FO feeding did not alter killing of L929 cells by macrophages, compared with feeding the LF diet. It is concluded that the type of fat in the diet affects macrophage composition and alters the ability of macrophages to produce cytotoxic and immunoregulatory mediators and to kill target tumour cells.     

Induction of nuclear lamins A/C in macrophages in in vitro cultures of rat bone marrow precursor cells and human blood monocytes, and in macrophages elicited in vivo by thioglycollate stimulation

Hemopoietic cells from blood and bone marrow of mammals usually do not express lamins A/C but only lamin B, and this feature distinguishes these cells from the vast majority of somatic cells of the adult animal, which reveal lamins A/C as well as lamin B. Here we have cultivated rat bone marrow precursor cells and human monocytes isolated from peripheral blood in tissue culture supplemented with certain growth factors. These conditions allow bone marrow precursor cells and monocytes to differentiate almost quantitatively into accessory cells and/or mature macrophages. The different cell types in the cultures can be identified both morphologically and by other assays. Antibodies specific for mouse A/C lamins, human A/C lamins, or B lamins have been used to define the lamin complement as a function of time in culture and of cell type. A dramatic increase in lamin A/C-positive cells was observed in the first 3 days of culture with both accessory cells and macrophages expressing lamins A/C as soon as such cell types could be identified. Parallel in vivo experiments showed that treatment with thioglycollate caused the percentage of lamin A/C-positive peritoneal macrophages to increase from 5 to 80% between Days 0 and 6.     

B1-B cells are the main antigen presenting cells in CpG-ODN-stimulated peritoneal exudate cells

Peritoneal exudate cells (PEC) have long been used as antigen presenting cells (APC), because they have been considered to contain mainly macrophages. However, it is still unclear specifically which cells of the peritoneal exudate function as APC. Herein, we focused on macrophages and B1-B cells of the PEC and examined their APC function and cytokine production. B1-B cells purified from PEC functioned effectively as APC after CpG-stimulation and mainly produced IL-10. In contrast, macrophages purified from PEC were not able to present incorporated antigens to T cells, despite the production of IL-12 and expression of co-stimulatory molecules after CpG stimulation. These results suggest that previously held ideas regarding the functions of the mixture of cells in the PEC need to re-evaluated. In summary, the antigen presenting function of PEC was mainly attributed to B1-B cells and immunoenhancing cytokine production was dominantly derived from peritoneal macrophages.     

The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5⁺ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.     

Burkholderia cenocepacia induces neutrophil necrosis in chronic granulomatous disease

Burkholderia cepacia complex is a life-threatening group of pathogens for patients with chronic granulomatous disease (CGD), whose phagocytes are unable to produce reactive oxygen species (ROS). Unlike other CGD pathogens, B. cepacia complex is particularly virulent, characteristically causing septicemia, and is the bacterial species responsible for most fatalities in these patients. We found that a nonmucoid Burkholderia cenocepacia (a predominant species in the B. cepacia complex) isolate was readily ingested by normal human neutrophils under nonopsonic conditions and promoted apoptosis in these cells. The proapoptotic effect was not due to secreted bacterial products, but was dependent on bacterial viability. Phagocytosis was associated with a robust production of ROS, and the apoptotic neutrophils could be effectively cleared by monocyte-derived macrophages. The proapoptotic effect of B. cenocepacia was independent of ROS production because neutrophils from CGD patients were rendered apoptotic to a similar degree as control cells after challenge. More importantly, neutrophils from CGD patients, but not from normal individuals, were rendered necrotic after phagocytosis of B. cenocepacia. The extreme virulence of B. cepacia complex bacteria in CGD, but not in immunocompetent hosts, could be due to its necrotic potential in the absence of ROS.     

Extracellular Vesicles from Leishmania-Infected Macrophages Confer an Anti-infection Cytokine-Production Profile to Na?ve Macrophages

Background

Extracellular vesicles (EVs) are structures with phospholipid bilayer membranes and 100–1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles.

Methodology/Principal Findings

In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50–300 nm-diameter extracellular vesicles (EVs). The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1β, and TNF-α.

Conclusions/Significance

EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1β and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.