Regulatory role of bone marrow in immunogenesis. III. Humoral factors stimulating and suppressing antibody formation produced by bone marrow cells in in vitro cultures

As revealed by the method of cultivation of bone marrow and spleen cells, separated by nucleopore membrane, in two-chamber bottles, the bone marrow cells were capable of producing humoral factor stimulating antibody genesis by the spleen cells. A direct contact of the bone marrow cells with the actively proliferating antigen-stimulated cells of the spleen led to production of a spleen humoral factor suppressing the antibody genesis by the spleen cells. The suppressive action of the bone marrow cells on the antibody genesis in the culture of the spleen cells was mediated through the suppression of the spleen cells proliferation; proliferation of the bone marrow cells is enhanced.     

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.     

Hypogammaglobulinemia in BLT Humanized Mice – An Animal Model of Primary Antibody Deficiency

Primary antibody deficiencies present clinically as reduced or absent plasma antibodies without another identified disorder that could explain the low immunoglobulin levels. Bone marrow-liver-thymus (BLT) humanized mice also exhibit primary antibody deficiency or hypogammaglobulinemia. Comprehensive characterization of B cell development and differentiation in BLT mice revealed other key parallels with primary immunodeficiency patients. We found that B cell ontogeny was normal in the bone marrow of BLT mice but observed an absence of switched memory B cells in the periphery. PC-KLH immunizations led to the presence of switched memory B cells in immunized BLT mice although plasma cells producing PC- or KLH- specific IgG were not detected in tissues. Overall, we have identified the following parallels between the humoral immune systems of primary antibody deficiency patients and those in BLT mice that make this in vivo model a robust and translational experimental platform for gaining a greater understanding of this heterogeneous array of humoral immunodeficiency disorders in humans: (i) hypogammaglobulinemia; (ii) normal B cell ontogeny in bone marrow; and (iii) poor antigen-specific IgG response to immunization. Furthermore, the development of strategies to overcome these humoral immune aberrations in BLT mice may in turn provide insights into the pathogenesis of some primary antibody deficiency patients which could lead to novel clinical interventions for improved humoral immune function.     

Properties of Educated T Cells for Rosette Formation and Cooperation with T Cells

IT has been shown that the humoral antibody response in mice to many antigens requires cooperation between thymus derived lymphocytes (T cells) and bone marrow derived lymphocytes (B cells)^1,2. The B cells are the direct precursors of antibody secreting cells and, although T cells react specifically with antigen, their role is unknown^2–6.     

Effect of "rasayanas", a herbal drug preparation on immune responses and its significance in cancer treatment.

Rasayanas are considered to be immunostimulating preparations used extensively in indigenous medical practice. However there are only very few reports to substantiate this claim, and this paper gives preliminary evidence for the potentiation of immunity by Rasayanas given to mice orally. Administration of Rasayanas were found to enhance the proliferation of spleen cells significantly especially in the presence of mitogen. Similar result was also seen with bone marrow cells; however mitogenic stimulation could not be observed. Esterase activity was found to be enhanced in bone marrow cells indicating increased maturation of cells of lymphoid linkage. Rasayanas also enhanced humoral immune response as seen from the increased number of antibody forming cells and circulating antibody titre. These results indicate the usefulness of Rasayana as immunostimulating agent.     

Mechanism of hybrid resistance. The role of a natural antibody in parental bone marrow cell rejection

Hybrid resistance (HR) to parental bone marrow growth is specifically directed against hemopoietic histocompatibility (Hh-1) Ag that are present in parental bone marrow cells (bmc). The mechanism of HR seems to be a multistep process. According to a model we proposed earlier, a T cell recognizes the Hh-1 Ag and stimulates a macrophage to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta activates a NK-like cell that specifically kills the parental bmc (effector phase). We have also described in a previous paper that serum from resistant F1 hybrids contains a humoral factor that seems to be involved in the effector phase of HR. In the present work, we study the role and the nature of this humoral factor. Our results show that this humoral factor: 1) is present in all resistant H-2Db heterozygous F1 hybrids we have tested but not in nonresistant H-2Db homozygous mice; 2) seems to recognize the Hh-1b Ag because it is absorbed on bmc from Hh-1b mice but not on bmc from Hh-1d and Hh-1- mice; and 3) is an IgG1 Ig (natural antibody). These results could help us to explain the specificity of HR at the effector phase by supposing that this natural antibody recognize the Hh-1 Ag and enable NK-like cells to kill parental bmc cells in Hh-1 specific manner.     

Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice

CD20 mAb-mediated B cell depletion is an effective treatment for B cell malignancies and some autoimmune diseases. However, the full effects of B cell depletion on natural, primary, and secondary Ab responses and the maintenance of Ag-specific serum Ig levels are largely unknown. The relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity also remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. Peritoneal B cell depletion reduced natural and Ag-induced IgM responses. Otherwise, CD20+ B cell depletion prevented humoral immune responses and class switching and depleted existing and adoptively transferred B cell memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow Ab-secreting plasma cell numbers. Coblockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the bone marrow. CD20+ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the bone marrow, with a significant decrease in Ag-specific serum IgG. Collectively, these results support previous claims that bone marrow plasma cells are intrinsically long-lived. Furthermore, these studies now demonstrate that mature and memory B cells are not required for maintaining bone marrow plasma cell numbers, but are required for repopulation of plasma cell-deficient bone marrow. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on preexisting Ab levels.     

Maintenance of serological memory

Long-lived plasma cells are key to maintaining long-term humoral immunity after infection or vaccination. Some vaccines and/or infections induce antibody levels that remain stable for the life of the individual. However, the mechanism whereby these long-lived plasma cells are maintained over long periods of time remains an open question. Furthermore, given a finite number of sustainable plasma cells within the bone marrow, it is also unclear how space for newly induced plasma cells is generated without compromising the pre-existing repertoire. Here we review the current understanding of these important issues.     

Genetics of resistance to the African trypanosomes. IV. Resistance of radiation chimeras to Trypanosoma rhodesiense infection

The cellular bases of resistance to the African trypanosomes were examined in inbred mice. As part of these studies, reciprocal bone marrow cell transplants were performed between H-2 compatible mice which differ in relative resistance to Trypanosoma brucei rhodesiense infection. Survival times, parasitemias and IgM antibody responses to the surface antigen of the infecting variant type were measured in these semiallogeneic bone marrow chimeras. Relatively resistant C57BL/10 mice, intermediate A.By mice, and least resistant C3H.SW mice that were reconstituted after lethal irradiation with syngeneic bone marrow cells displayed resistance and immunity characteristic of the homologous donor strain. When C57BL/10 mice were reconstituted with C3H.SW mouse bone marrow cells they retained the ability to produce antibodies to trypanosome surface antigen but the antibody titers were significantly reduced. Control of parasitemia and mean survival time were reduced in these chimeras, but differed significantly from C3H.SW mice. A.By mice that received cells from C57BL/10 donors exhibited antibody responses and survival times similar to the C57BL/10 mice. Survival times of A.By mice given syngeneic cells or C3H.SW cells were the same, but the antibody responses of A.By mice given C3H.SW cells were lower than those of A.By mice given syngeneic cells. C3H.SW mice reconstituted with C57BL/10 bone marrow cells were capable of making antibodies and controlling parasitemia, in marked contrast to the absence of such responses in C3H.SW mice reconstituted with syngeneic cells. Survival times, however, were indistinguishable from those of C3H.SW mice given syngeneic cells. Thus, resistance to T. b. rhodesiense was shown for the first time to depend on donor bone marrow derived cells as well as upon radiation-resistant cells/factors associated with host genetic background. Also, parasite-specific IgM antibody responses seem to be regulated by a mechanism which does not depend on bone marrow derived cells alone, and the presence of such immune responses is not linked to survival time.     

The xid mutation affects hemopoiesis in long term cultures of murine bone marrow

Cells of the humoral immune system are particularly affected by a mutation at the X chromosome linked immunodeficiency disease (xid)locus. Although B cells are made in normal numbers, they fail to become phenotypically and functionally diverse. Consequently, poor antibody responses are mounted to certain types of Ag. There have been some indications that other types of hemopoietic cells may be influenced by the mutation and development of the humoral immune system is unusually dependent on the presence of T lymphocytes. We now describe an analysis of the lympho-hemopoietic environment studied with long term bone marrow cultures. Contrary to expectations, cultures initiated with cells from homozygous female or hemizygous male mice with the mutant allele established more quickly than normal. The accelerated initial growth pattern was clearly linked to the xid mutation. Artificial mixtures of marrow exhibited intermediate growth kinetics. Experiments with H-2 congenic and T6 chromosome marked cells did not reveal an intrinsic dominance of growth in nonadherent xid cells. Similar results were obtained with culture conditions which favored production of myeloid or lymphoid cells. These findings would be consistent with subtle changes in the bone marrow microenvironment resulting from the xid mutation. The pedigree of the mouse strains had a significant influence on lymphopoiesis in long term bone marrow cultures. Lymphocytes of BALB/c origin dominated over CBA/H background cells in cultures established from mixtures of the two, but this did not correlate with any functional deficiency in CBA/H stromal cells. In fact, establishment of an adherent layer was a rate-limiting step in initiating long term cultures and this could be achieved with a low dose inoculum of CBA/H marrow. Even more dramatic effects were found in hemopoietic cells from doubly defective C3H.nu/nu-xid mice. The bone marrow of these athymic animals contained normal numbers of granulocyte/macrophage progenitors. However, lymphoid cultures could not be reproducibly established with their cells and myelopoiesis was never observed in vitro. The relatively simple conditions which pertain in culture make it possible to appreciate effects of mutations and pedigree on hemopoiesis which are unremarkable in intact animals.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

The role of the bone marrow in forming an immunological memory to Staphylococcus

The dynamic study of antibody formation in the bone marrow tissue of mice immunized with corpuscular staphylococcal antigen was made. A single intravenous injection of this antigen to the animals produced no essential changes in the number of antibody-forming cells in the bone marrow, their repeated immunization led to the involvement of the bone marrow in the anamnetic reaction, which was manifested by the accumulation of a great number of plasmocytes synthesizing antibodies to staphylococci in bone marrow tissue. The relative contribution of the bone marrow to systemic antibody formation essentially increased at later stages of secondary immune response. The possible biological significance of the regularities and the practical importance of the data revealed in this investigation are discussed.     

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.     

Differential effects of abrin on normal and tumor cells

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.     

The effect of haemopoietic stem cell proliferation on the humoral immune response in mice

A study was made of the effect of humoral factors, isolated from bone marrow cell (BMC) supernatant fluid and capable of modifying CFU-S proliferation, on the generation of IgM plaque-forming cells (PFC) against sheep red blood cells (SRBC) in mice after adoptive transfer. Adoptive transfer of BMC, preincubated with the humoral factor RBME-III, which stimulates CFU-S proliferation, was shown to suppress the splenic PFC generation in recipients; treatment of BMC with a further factor NBME-IV, which inhibits CFU-S proliferation, was followed by augmentation of PFC generation. Similar effects were obtained while studying the IgM PFC generation in the bone marrow of mice after secondary immunization when relevant factors were injected, in vivo, 24 hr following primary immunization. The results of adoptive transfer experiments indicate that populations of T- and B-cells are not the targets for the action of CFU-S proliferation regulatory factors. These factors are shown to modulate the erythroid differentiation of CFU-S. The possibility of quantitative modification of immune response parameters with the help of bone marrow factors that influence the proliferation and differentiation of CFU-S is discussed.     

The effect of haemopoietic stem cell proliferation on the humoral immune response in mice

Abstract A study was made of the effect of humoral factors, isolated from bone marrow cell (BMC) supernatant fluid and capable of modifying CFU-S proliferation, on the generation of IgM plaque-forming cells (PFC) against sheep red blood cells (SRBC) in mice after adoptive transfer. Adoptive transfer of BMC, preincubated with the humoral factor RBME-III, which stimulates CFU-S proliferation, was shown to suppress the splenic PFC generation in recipients; treatment of BMC with a further factor NBME-IV, which inhibits CFU-S proliferation, was followed by augmentation of PFC generation. Similar effects were obtained while studying the IgM PFC generation in the bone marrow of mice after secondary immunization when relevant factors were injected, in vivo , 24 hr following primary immunization. The results of adoptive transfer experiments indicate that populations of T- and B-cells are not the targets for the action of CFU-S proliferation regulatory factors. These factors are shown to modulate the erythroid differentiation of CFU-S. The possibility of quantitative modification of immune response parameters with the help of bone marrow factors that influence the proliferation and differentiation of CFU-S is discussed.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

Studies of the immunological capacity of germ-free mouse radiation chimeras: I. Chimerism and humoral immune response

The genetic origin of both the functional lymphoid cell and progenitor cell populations of germ-free mouse radiation chimeras was assessed by indirect immunofluorescence (IIF), anti-H-2 cytotoxicity, and survival of lethally x-irradiated secondary recipients of chimera cell populations. These studies demonstrated that germ free C3H/He mice given 1000 R and 10⁷ DBA/2 bone marrow cells express H-2 antigens on their lymphoid and bone marrow cell populations characteristic of the DBA/2 donor. These cells persist for at least 14 months postirradiation and bone marrow transplantation. However, these allogeneic mouse radiation chimeras have a reduced humoral immune response to sheep erythrocytes (SRBC). This decreased humoral immune capacity as assessed by kinetic studies of the spleen plaque-forming cell (PFC) response is present throughout the life span of the chimera. The γ₁ PFC response shows extreme depression. The reduced humoral immune responsiveness to the thymusdependent SRBC antigen is considered to be due to the absence or malfunctioning of a thymocyte population.     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.     

Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.