Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus

We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.     

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to gamma-irradiation.

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. We have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to gamma-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-gamma (rmIFN-gamma) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by gamma-irradiation. Concomitant priming of gamma-irradiated J774 M phi with rmIFN-gamma increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC. Irradiated J774 cells will also provide a homogenous, stably primed cell type in which to examine the mechanism(s) of cytotoxicity employed by tumoricidal M phi.     

Efficient natural defense mechanisms against Listeria monocytogenes in T and B cell-deficient allogeneic bone marrow radiation chimeras. Preactivated macrophages are the main effector cells in an early phase after bone marrow transfer

Radiation chimeras in the early phase after bone marrow transplantation are a good model to study the efficiency of the body's nonspecific defense system represented by macrophages (M phi), polymorphonuclear cells (PMN), and NK cells. These cell types are present in large numbers in spleen and liver at that time, whereas the specific immune system represented by T and B cells is functionally deficient. We previously reported enhanced activities in vitro of M phi (and PMN) from recipient animals in an early phase after allogeneic bone marrow transfer. We here demonstrate that these activities result in enhanced spontaneous resistance against Listeria monocytogenes in vivo: CFU of L. monocytogenes in spleen and liver 48 h after infection were about 1 or 2 to 4 log steps less than in untreated control mice of donor or host haplotype. This enhanced resistance decreased over the 4-mo period after marrow transfer. Preactivated M phi were identified as the most important effector cells. Isolated from spleen and peritoneal cavity, they performed enhanced killing of phagocytosed Listeria. Such preactivated M phi occurred in recipient animals after transfer of allogeneic but not of syngeneic bone marrow. The precise mechanism of M phi activation in the allogeneic radiation chimera in the complete absence of any detectable T cell function is not clear at present. However, these preactivated M phi display an important protective effect against L. monocytogenes: chimeras could eliminate Listeria without acquisition of positive delayed-type sensitivity when infected with 10(3) bacteria. An inoculum of 5 . 10(3) L. monocytogenes resulted either in prolonged survival compared with normal mice of the recipient haplotype or in definitive survival accompanied by a positive delayed-type sensitivity. We concluded that enhanced nonspecific immune functions can in part compensate for the defective specific immune system after bone marrow transfer.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pM phi-mediated cytotoxicity and pM phi protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pM phi to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pM phi were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.     

Modulation of antigen-induced T cell proliferation by alpha 2M-trypsin complexes

Proteinase-complexed alpha 2-macroglobulin (alpha 2M) could be shown to interfere with T cell proliferation in response to antigen presented by autologous antigen-pulsed monocytes (M phi) (antigen-induced M phi-T cell interaction, MTI). Addition of alpha 2M-trypsin (alpha 2M X T) complexes to cultures of T cells and antigen-pulsed M phi led to a dose-dependent decrease of T cell proliferation (up to 91% inhibition of the T cell response), whereas the same concentrations of free (native) alpha 2M had no effect on antigen-induced MTI. The observed interference with MTI could be attributed to residual enzymic activity of the alpha 2M X T complex. Addition of aprotinin, a low Mr protein proteinase inhibitor able to penetrate to the enzyme entrapped within the alpha 2M molecule and thus bind to and inactivate the enzyme's active site, resulted in a reversal of the alpha 2M X T-induced biological effect. Inactivation of the enzyme's active site within alpha 2M X T was monitored by a decrease in the hydrolytic activity of the complex. Kinetic studies (addition of alpha 2M X T 24 to 48 hr after culture onset was shown to be still inhibitory) indicated an effect at the level of the T cell or its mediators, but an overnight incubation of T cells with alpha 2M X T did not alter these cells' capacity to proliferate in response to an antigenic stimulus. An additional effect of alpha 2M X T on the antigen-presenting cell cannot be ruled out at present. However, alpha 2M X T did not alter the percentage of monocytes expressing HLA-DR, -DP, and -DQ or interfere with interleukin 1 release if added to M phi at concentrations that significantly inhibited MTI. Furthermore, incubation of M phi with alpha 2M X T for 1 hr before antigen pulsing had no effect on the M phi antigen presenting capacity.     

Heterogeneity of human monocyte subsets in the promotion of B cell colonies and the role of interleukin 1

The abilities of human peripheral blood mononuclear-phagocyte (M phi) subpopulations and of interleukin 1 (IL 1) to support human B cell colony formation in semisolid cultures stimulated by staph protein A were analyzed. Human M phi subsets enriched for complement receptors (CR) effectively functioned as accessory cells supporting colony growth, whereas the responses obtained with CR-depleted M phi were 4.6-fold less. In experiments analyzing IL 1 production, CR-enriched M phi secreted four to 12 fold greater amounts of basal and stimulated IL 1 than CR-depleted M phi. Also, the addition of IL 1 to CR-depleted M phi resulted in a fourfold increase of colony numbers. The responses of cultures containing CR-depleted M phi plus IL 1, however, remained 30% less than those observed for cultures supplemented with CR-enriched M phi. Other studies showed that IL 1 was unable to substitute for M phi; the responses of cultures containing IL 1 and B cells were reduced 10-fold compared to cultures supplemented with autologous M phi. These findings indicate that human M phi subsets exist that differ in their ability to function as accessory cells. Although IL 1 can collaborate with certain M phi subsets to restore their accessory cell function, it cannot replace intact M phi. Thus, it is possible that other monokines or lymphokines play a role in M phi accessory cell function.     

Differential abilities of human peripheral blood monocytes quantitatively or qualitatively differing in HLA-DR and HLA-DS expression to support B cell activation in liquid and semisolid cultures

The present investigation was performed to determine whether the activation of human B cells by Staphylococcal protein A (SpA) in liquid and semi-solid cultures might be dependent on distinct subsets of peripheral blood mononuclear-phagocytes (M phi) defined by the expression of HLA-DR and HLA-DS determinants. Highly pure HLA-DR- M phi functioned as effectively as HLA-DR+ MO in supporting B cell liquid proliferative responses when SpA was continuously present in cultures. However, HLA-DR+ M phi were two to three times more effective than HLA-DR- M phi in promoting B cell proliferative responses when either M phi or B cells were pulsed with SpA and were then cultured without supplemental SpA. Similarly, B cell activation in semisolid cultures was crucially dependent on HLA-DR+ M phi because colony responses were reduced fivefold in the presence of M phi expressing low/intermediate HLA-DR levels compared to M phi-containing cells with high HLA-DR levels. HLA-DS- M phi isolated by two different techniques were more effective than HLA-DS+ M phi in supporting both liquid proliferative and colony responses of B cells. Flow microcytofluorometry analysis of the dual expression of HLA-DR and HLA-DS on highly pure HLA-DR- M phi and HLA-DR+ M phi revealed that both HLA-DR- and HLA-DR+ M phi expressed low levels of HLA-DS. Importantly, the expression of HLA-DS on HLA-DR- M phi was bimodal, with an HLA-DR-, DS+ subset and an HLA-DR-, DS-subset being present. Other experiments supported the conclusions that the differential abilities of the HLA-DR-, -DS-defined subsets of M phi to support B cell activation did not represent M phi suppressive effects or differences in IL 1 production. Collectively, these results indicate that B cell activation can be directly supported by M phi whose predominant phenotype is HLA-DR+, -DS-. Thus, the accessory cell pathway of B cell activation described here is distinct from the pathway known to be required for T cell responsiveness, and could serve to provide early alternative or ancillary signals for triggering B cells.     

Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major

Macrophages are the major target cell population of the obligate intracellular parasites LEISHMANIA: Although polymorphonuclear neutrophil granulocytes (PMN) are able to internalize Leishmania promastigotes, these cells have not been considered to date as host cells for the parasites, primarily due to their short life span. In vitro coincubation experiments were conducted to investigate whether Leishmania can modify the spontaneous apoptosis of human PMN. Coincubation of PMN with Leishmania major promastigotes resulted in a significant decrease in the ratio of apoptotic neutrophils as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low m.w. DNA fragments, and annexin V staining. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in PMN. The inhibition of PMN apoptosis depended on viable parasites because killed Leishmania or a lysate of the parasites did not have antiapoptotic effect. L. major did not block, but rather delayed the programmed cell death of neutrophils by approximately 24 h. The antiapoptotic effect of the parasites could not be transferred by the supernatants, despite secretion of IL-8 by PMN upon coculture with L. major. In vivo, intact parasites were found intracellularly in PMN collected from the skin of mice 3 days after s.c. infection. This finding strongly suggests that infection with Leishmania prolongs the survival time of neutrophils also in vivo. These data indicate that Leishmania induce an increased survival of neutrophil granulocytes both in vitro and in vivo.     

Modulation of Ia-like antigen expression and antigen-presenting activity of human monocytes by endotoxin and zymosan A

The environmental agents E. coli endotoxin and zymosan A modulated antigen-specific T cell proliferation in vitro, assessed by 3H-TdR uptake. In the continual presence of these agents, human mononuclear leukocyte responses to the antigens tuberculin PPD, Candida albicans, and mumps were significantly reduced. Treatment of adherent cell-depleted T cells with the agents did not affect their subsequent reactivity to soluble antigens in the presence of normal M phi. However, cultures consisting of pretreated M phi, normal T cells, and soluble antigen gave responses that were only 7 to 38% of control values, indicating that the function of the antigen-presenting cell, not the T cell, was inhibited. This effect was observed only when treatment with endotoxin or zymosan A preceded antigen stimulation by at least 24 hr, suggesting that a gradual inhibition of antigen presentation had occurred. When various ratios of normal antigen-pulsed and agent-treated M phi were cultured with normal T cells, antigen-specific responses were not significantly different from control cultures; this indicated that M phi-mediated suppression was not involved. It did not appear that the inhibition was due to enhanced antigen degradation by the treated M phi because responses were not reconstituted in the presence of excess antigen. After endotoxin or zymosan A treatment of the M phi population the proportion of Ia+ cells was reduced significantly, and surface expression of Ia antigen correlated with the ability of the cell population to present antigens to immune T cells. This suggested that endotoxin and zymosan A induce a loss of surface Ia antigen on antigen-presenting cells that inhibits immune T cell activation.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamics of cytochemically distinct subpopulations of macrophages in elicited rat peritoneal exudates

Qualitative and quantitative changes in rat peritoneal macrophage (M phi) subpopulations, differing in their ultrastructural peroxidatic staining characteristics were followed over the course of a thioglycollate (TG) broth-induced inflammatory response. In addition, selected functional features of the normal steady-state and 4-day TG-induced populations of M phi were compared. The steady-state population consisted primarily of M phi with peroxidatic staining limited to the nuclear envelope (NE) and rough endoplasmic reticulum (RER); such cells are called resident M phi. Within hours of TG injection, there was an influx of monocyte-derived exudate M phi, the number of which reached a maximum, by 24 hr. During the next 24 hr, the proportion of exudate M phi decreased with a concomitant increase in peroxidatic activity (PA)-negative M phi. These two cell types continued to predominate for the next 48 hr during which there was a gradual increase in resident M phi and so-called "exudate-resident" M phi, the latter of which exhibits both exudate and resident PA patterns. Thus, the 4-day TG-induced population consisted of four cytochemically distinct M phi subpopulations: approximately 50% PA-negative M phi, approximately 25% exudate M phi, approximately 15% resident M phi, and approximately 10% exudate-resident M phi. Differences in Fc receptors and complement receptors 1 and 3 were noted between the two populations in the presence of progenitors that give rise to colonies of M phi in liquid culture in response to murine-derived colony-stimulating factor 1. The implications of these results in regard to the origin(s) of M phi diversity are discussed.     

Fibroblast-secreted macrophage colony-stimulating factor is responsible for generation of biphenotypic B/macrophage cells from a subset of mouse B lymphocytes.

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/M phi) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/M phi cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/M phi cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/M phi cells. The transition from B lymphocyte to B/M phi cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/M phi cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-gamma in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/M phi cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/M phi cells.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Pre-existing glomerular immune complexes induce polymorphonuclear cell recruitment through an Fc receptor-dependent respiratory burst: potential role in the perpetuation of immune nephritis

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.     

The role of interleukin 1 in human B cell activation: inhibition of B cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen

The role of factors released by monocytes (M phi) in the activation of human B lymphocytes was examined by studying the effect of an antiserum against human leukocytic pyrogen (LP) on mitogen-stimulated B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) by peripheral blood mononuclear cells (PBM). Antiserum against LP was obtained from rabbits immunized with LP-containing human M phi supernatants. The globulin fraction of this antiserum inhibited pokeweed mitogen- (PWM) stimulated B cell proliferation and the generation of ISC in a concentration-dependent manner, with 50% inhibition of responsiveness observed with 10 micrograms/ml. By contrast, PWM-induced T cell [3H]thymidine incorporation was not inhibited by concentrations of anti-LP as great as 2000 micrograms/ml. The F(ab')2 fraction of anti-LP also inhibited the generation of ISC in response to both PWM and formalinized Staphylococcus aureus, but required 50 micrograms/ml to achieve 50% inhibition. Anti-LP inhibited the generation of ISC only if present during the first 24 hr of a 6 to 7-day incubation; later addition was not inhibitory. Inhibition was more marked in cultures partially depleted of M phi than in whole PBM cultures. Whereas absorption of the anti-LP with PBM failed to remove the capacity to inhibit the generation of ISC, anti-LP-mediated inhibition of responsiveness could be reversed by the addition of crude M phi culture supernatants or a variety of highly purified interleukin 1 (IL 1) preparations, but not by T cell supernatants. These results indicate anti-LP inhibits human B cell activation by removing the requisite M phi-derived factor IL 1 and also confirm that IL 1 plays an essential role in B cell proliferation and the generation of ISC in man.     

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact

We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM. © 1994 Wiley-Liss, Inc.     

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.     

Reduced macrophage recruitment,proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation

Kidney tubular epithelial cell (TEC) death may be dependent on the number and activation state of macrophages (M phi) during inflammation. Our prior studies indicate that activated M phi release soluble mediators that incite TEC death, and reducing intrarenal M phi during kidney disease diminishes TEC apoptosis. CSF-1 is required for M phi proliferation and survival. We hypothesized that in the absence of CSF-1, M phi-mediated TEC apoptosis would be prevented during renal inflammation. To test this hypothesis, we evaluated renal inflammation during unilateral ureter obstruction in CSF-1-deficient (Csf1(op)/Csf1(op)) mice. We detected fewer M phi and T cells and less apoptotic TEC in the obstructed kidneys of Csf1(op)/Csf1(op) mice compared with wild-type (WT) mice. The decrease in intrarenal M phi resulted from diminished recruitment and proliferation, not enhanced apoptosis. CSF-1 enhanced M phi activation. There were far fewer activated (CD69, CD23, Ia, surface expression) M phi in obstructed CSF-1-deficient compared with WT obstructed kidneys. Similarly, bone marrow M phi preincubated with anti-CSF-1 receptor Ab or anti-CSF-1 neutralizing Ab were resistant to LPS- and IFN-gamma-induced activation. We detected fewer apoptotic-inducing molecules (reactive oxygen species, TNF-alpha, inducible NO synthase) in 1) M phi propagated from obstructed Csf1(op)/Csf1(op) compared with WT kidneys, and 2) WT bone marrow M phi blocked with anti-CSF-1 receptor or anti-CSF-1 Ab compared with the isotype control. Furthermore, blocking CSF-1 or the CSF-1 receptor induced less TEC apoptosis than the isotype control. We suggest that during renal inflammation, CSF-1 mediates M phi recruitment, proliferation, activation, and, in turn, TEC apoptosis.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.