Bradykinin sensitization of CSF-1-responsive murine mononuclear phagocyte precursors to prostaglandin E

Bradykinin (BK) inhibited clonal proliferation of CSF-1-stimulated mononuclear phagocyte precursors derived from murine marrow. This inhibitory effect of BK was restricted to the subpopulation of precursors that required two signals, CSF-1 and LPS, for clonal proliferation. No effect was observed on stimulated colony formation by precursors that responded solely to the addition of CSF-1. Inhibition of colony formation by the two signal-dependent precursors required the presence of adherent marrow cells and was mediated by endogenously produced PG. Although evidence was obtained indicating that BK augmented PG production by adherent cells, an additional effect of BK appeared to be a significant sensitization of the two signal-dependent precursors to inhibition by PGE.     

Bradykinin sensitization of colony-stimulating factor-1-responsive murine marrow progenitors to prostaglandin E. A property of the amino-terminal tetrapeptide fragment

The active sequence in bradykinin (BK) responsible for PGE-aided inhibition of CSF-1-stimulated clonal proliferation of murine mononuclear phagocyte progenitors was determined. In total marrow cultures, BK and (D-Phe7)-BK, a specific BK antagonist, inhibited colony formation by CSF-1 responsive precursors that require two signals, CSF and LPS, for clonal proliferation. (Lys1)-BK, an inactive BK analogue with Lys substituted for the amino-terminal Arg, was inactive. Arg-Pro-Pro-Gly, the amino-terminal tetrapeptide fragment of BK, was fully capable, on a molar basis, of replacing either BK or (D-Phe7)-BK as an inhibitor. Bk, (D-Phe7)-BK, and Arg-Pro-Pro-Gly were not inhibitory for colony formation in cultures containing indomethacin or in cultures depleted of adherent marrow cells. However, in these cultures addition of 10(-9) M PGE2 fully restored inhibition of two-signal-dependent colony formation. PGE2-dependent inhibition by the three peptides was equivalent on a molar basis indicating that Arg-Pro-Pro-Gly contains the sequence responsible for this inhibitory effect of BK and is sufficient to exert PGE-dependent inhibition of two-signal-dependent colony formation. The two-signal-dependent progenitors appear to be in transition to CSF competence suggesting that BK and PGE produced in an hematopoietic environment may act together to limit the production of new macrophages by inhibiting progenitors in transition to CSF competence.     

Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect

The inflammatory neuropeptide substance P acted as a costimulant for macrophage CSF-1-induced clonal proliferation of murine marrow-derived two signal-dependent mononuclear phagocyte progenitors. Substance P had no effect on clonal proliferation by progenitors responding solely to CSF-1. Substance P fragment 2-11 had no costimulatory activity; however, SP fragment 1-4 retained the full activity of the parent undecapeptide. Fragment 1-4 (ARG-PRO-LYS-PRO), a peptide containing a PRO residue between two positive charges, is a tuftsin-like (THR-LYS-PRO-ARG) tetrapeptide, and tuftsin exerted an identical costimulatory effect. Substance P, SP:1-4, and tuftsin were optimally effective as costimulants at 10(-7) to 10(-6) M. (ALA1)-tuftsin, an inhibitory analog of tuftsin, was a potent negative regulator of two signal-dependent colony formation. (ALA1)-tuftsin at concentrations less than or equal to 10(-9) M exerted dose-dependent inhibition of the positive effects of optimal concentrations of all of the co-stimulants tested, including bacterial LPS. The inhibitory tetrapeptide was equivalent in activity to ferritin, an established inhibitor of two signal-dependent colony formation. The results indicated that SP may influence myelopoiesis in addition to its other inflammatory and immunopotentiating properties. In addition, a potentially valuable modulator of SP and LPS responses in this system, (ALA1)-tuftsin, was identified.     

Effect of Anti-Ia Serum In Vitro on Lipopolysaccharide-Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [³H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed.     

The expression of Ia antigenic determinants on macrophages required for the in vitro antibody response.

A subpopulation of antigen-presenting macrophages required for an in vitro antibody response to burro erythrocytes was deleted by pretreating the splenic macrophages with anti-Ia serum and complement (C). The in vitro response of the macrophage depleted T-B cell population could not be restored by the addition of macrophages resistant to anti-Ia antibodies and C (Ia-). The response of Ia- macrophages and the macrophage-depleted T-B cells was only reconstituted by the addition of Ia+ macrophages. Macrophages pretreated with anti-Ia antibodies restricted to react with determinants of one I subregion could not support the in vitro antibody response when added to cultures whose macrophages were pretreated with anti-Ia serum and C specific for the I-J subregion. These results confirmed that Ia determinants of the I-A, the I-E, and the I-C subregions were all expressed on the I-J+ macrophage required for an in vitro antibody response.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

Subpopulations of the T4+ inducer T cell subset in man: evidence for an amplifier population preferentially expressing Ia antigen upon activation

Prior studies demonstrated that Ia molecules were expressed on a fraction of human peripheral T4+ inducer cells upon stimulation by soluble antigen. In the present study, we utilized a fluorescence-activated cell sorter to separate antigen-activated T4+,Ia+ and T4+,Ia- populations and characterized their function. It was found that the T4+,Ia+ population contained the majority of proliferating T cells as assessed by tritiated thymidine incorporation. This proliferation largely appeared to be nonspecific. Despite macrophage repletion, elimination of the Ia+ subset of T cells with monoclonal anti-Ia antibody and complement treatment equally diminished subsequent proliferation to both the triggering antigen and an unrelated antigen. Moreover, the antigen-induced Ia+ subset of T cells alone produced a nonspecific helper factor, LMF. In contrast, the the T4+,Ia- population showed minimal proliferation to soluble antigen and did not generate LMF. Nevertheless, both T4+,Ia+ and T4+,Ia- inducer T cells were required to generate maximal immunoglobulin production by B cells in an antigen-driven system. We conclude that the human T4+ inducer T cell subset is comprised of at least 2 functionally distinct subpopulations, which are capable of acting in a synergistic fashion to provide help to B cells.     

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis

Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.     

The opposing actions in vivo on murine myelopoiesis of purified preparations of lactoferrin and the colony stimulating factors

The actions of purified iron-saturated human lactoferrin (LF), purified preparations of human MiaPaCa colony stimulating factor-1 (CSF-1), and recombinant murine interleukin-3 (IL-3) were evaluated in vivo in mice. Studies in vitro were compared at lowered (5%), as well as at normal incubator (20%), oxygen (O2) tension because of the potentially greater physiologic relevance of in vitro studies performed at lowered O2 tension. The results demonstrate that 1) increased release of granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro from pokeweed mitogen stimulated mouse spleen cells and from human mononuclear blood cells occurred at lowered O2 tension, and that human mononuclear blood leukocytes were more sensitive to the LF-induced suppression of GM-CSF release when cells were cultured at 5%, compared to 20%, O2 tension; 2) LF administered intravenously (IV) to mice pretreated with sublethal intraperitoneal dosages of Cytoxan decreased the cycling status of marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E-2 and BFU-E-1) and multipotential (CFU-GEMM) progenitor cells and the absolute numbers of these progenitors; these effects were most noticeable if care was taken to deplete endotoxin from the LF samples prior to testing LF in vivo and if the control medium was endotoxin free; 3) endotoxin-depleted LF decreased the cycling status of marrow and spleen CFU-GM, BFU-E, and CFU-GEMM and the numbers of these progenitors in the marrows of mice previously untreated with Cytoxan; these effects were most apparent when assessment of progenitor cells and their cycling rates were evaluated in vitro at lowered (5%) O2 tension; 4) purified natural human CSF-1 increased the absolute numbers of marrow CFU-GM and the cycling status of marrow CFU-GM and CFU-GEMM in mice pretreated with LF; and 5) purified recombinant murine IL-3 stimulated proliferation of day 8 and day 12 CFU-S (colony forming unit-spleen) in mice not previously treated with Cytoxan. These results substantiate the in vivo myelosuppressive effects of LF on CFU-GM and extend these effects to erythroid and multipotential progenitor cells, provide evidence that human CSF-1 has an in vivo action in mice, and confirm the studies of others showing that IL-3 stimulates the proliferation of CFU-S in vivo.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. Populations of adherent bone marrow-derived macrophages (BMM) devoid of CSF-1 producing cells were used to study regulation by CSF-1 of macrophage entry into S phase. More than 95% of BMM possess the CSF-1 receptor. It was shown that 93-98% of BMM are cycling (S phase 8-9 hr, doubling time 24-28 hr) when cultured in the presence of CSF-1. BMM incubated with 15% FCS in the absence of CSF-1 or in the presence of CSF-1 concentrations inducing survival without proliferation enter a quiescent state. This state is characterized by a reduction in the synthesis of DNA (98%), total protein (35%), ribosomal protein (76%), and histone (96%) compared with the synthetic rate of these components in exponentially growing cells. Addition of CSF-1 to BMM rendered quiescent by removal of CSF-1 stimulated entry into S phase with a lag period of approximately 12 h. This lag period is reduced to 8 hr in BMM made quiescent at concentrations of CSF-1 inducing survival without proliferation, an effect which may be related to the expected higher protein content of these cells (Tushinski and Stanley, J. Cell. Physiol., 116:67-75). Neutralization of CSF-1 by antibody at different times during the lag period indicates that CSF-1 is required for almost the entire lag period for the entry of any cells into S phase. In BMM rendered quiescent by removal of both serum and CSF-1, purified CSF-1 without serum stimulated entry of cells into S phase, whereas serum alone was ineffective. The results are consistent with a primary regulatory role of CSF-1 in mononuclear phagocyte proliferation, survival, and function.     

Requirement for an Ia-bearing accessory cell in Con A-induced T cell proliferation.

Pretreatment of murine lymphoid cells with anti-Ia and C abrogated the proliferative response of these cells to Con A, but not to PHA. Reconstitution experiments demonstrated that T cell-enriched populations failed to restore Con A responsiveness and that T cell-depleted populations were more effective in restoring responsiveness to Con A. In particular, a population of 1000 R resistant, glass-adherent, non-T spleen cells was capable of completely restoring responsiveness to Con A when added in numbers as low as 4% of cultured cells. These splenic adherent cells were found to express Ia determinants encoded by at least two genes: one in I-A and the other in I-B, I-J, and/or I-E/C, and it was demonstrated that determinants encoded in these two regions were expressed on the same cell. These results demonstrate that non-T accessory cells may be the Ia+ cells entirely responsible for the anti-Ia and C-induced abrogation of T cell proliferative responses to Con A.     

Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.     

A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation.

A murine T lymphocyte proliferation assay that used antigen-primed lymph node T cells, was antigen specific, and required exogenous accessory cells was used to characterize the accessory cells that supported proliferation. These cells were Thy 1.2 negative, radioresistant, glass-adherent, and were functional only if alive. The accessory cell function of spleen adherent cells was much greater than that of peritoneal cells. Also, the accessory cell function of spleen adherent cells was proportional to the length of time such cells were incubated with antigen and very small numbers of such cells provided accessory cell function. Cytotoxic studies with subregion-restricted anti-Ia antibodies and complement indicated that accessory cell function resided in a subpopulation of spleen adherent cells that bore both I-A and I-E or C subregion antigens. The function of such cells was not related to a selective ability (vs other spleen adherent cells) to take up antigen. These data indicate that antigen-specific stimulation of T lymphocyte proliferation requires at least one specific subpopulation of spleen adherent cells that can be phenotypically identified by its expression of Ia antigens and are consistent with the possibility that Ia antigens may be Ir gene products.     

Increased Qa-m7 antigen expression is characteristic of primitive hemopoietic progenitors in regenerating marrow

Developmentally early murine hemopoietic progenitor cells of high proliferative potential (HPP-CFC), which are detectable in clonal agar culture in the presence of the lineage-specific hemopoietic growth factor, colony-stimulating factor-1 (CSF-1) plus hemopoietin-1 (H-1), or interleukin 3 (IL 3), express relatively high levels of the Qa-m7 antigenic determinant. This determinant is progressively lost during differentiation, and the more committed progenitors which grow in the presence of CSF-1 alone are essentially devoid of Qa-m7. Significant increases in both the proportion of Qa-m7-positive myeloid cells and the level of Qa-m7 antigen expression have been observed in bone marrow cells regenerating after the administration of the cytotoxic agent 5-fluorouracil (5-FU). By exploiting this increase in Qa-m7 antigen expression during regeneration and the HPP-CFC-sparing properties of 5-FU, we have been able to enrich HPP-CFC from marrows 8 days post-5-FU treatment (FU8d) to purities of greater than 20%. Furthermore, discontinuous gradient centrifugation and fluorescence-activated cell sorting of FU8d bone marrow cells on the basis of their light-scattering properties and Qa-m7 expression has unmasked a further subset of HPP-CFC which strictly requires the combined stimulus of three hemopoietic growth factors (H-1, IL 3, and CSF-1) for clonal growth. These highly enriched subsets of HPP-CFC are either identical to or co-fractionate with transplantable multipotential hemopoietic progenitors capable of reconstituting the hemopoietic system of lethally irradiated mice. Up to one in three cells in these highly enriched fractions is an HPP-CFC, and up to one in two cells may be CFU-S assayed 13 days post-transplantation. In addition, these fractions contain progenitors capable of reconstituting the platelet, erythroid, and myeloid compartments of the marrow.     

Synergistic hematopoietic growth factors

A number of growth factors acting on hematopoietic stem cells have now been purified and characterized. These include erythropoietin, granulocyte-macrophage colony-stimulating activity (GM-CSA), granulocyte colony-stimulating activity and colony-stimulating factor-1 (CSF-1). Factors which act in concert with these defined factors and appear to act relatively early in the hematopoietic stem cell lineage are currently under study. Interleukin 3 appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. Interleukin 3 acts in concert with at least CSF-1 and erythropoietin to enhance their effect on stem cell proliferation and differentiation. A new class of hematopoietic growth factor activities termed synergizing activities also exist. These activities appear to have no intrinsic capacity to stimulate hematopoietic colony formation by themselves but enhance the effects of other differentiating hormones such as GM-CSA and CSF-1. Activities which appear to represent synergizing activities have now been found to evolve from a human bladder carcinoma line, a cell line derived from murine marrow adherent cells and normal murine marrow and thymic cells. These activities may act on very primitive hematopoietic progenitors to allow them to express receptors to various differentiating hormones or alternatively they may act as commitment factors in a commitment-progression model of stem cell regulation.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

Inhibition of the signaling pathways for macrophage proliferation by cyclic AMP. Lack of effect on early responses to colony stimulating factor-1.

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in quiescent murine bone marrow-derived macrophages (BMM). CSF-1 action has been shown to involve activation of the CSF-1 receptor kinase. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (PMA), is itself weakly mitogenic and synergises with CSF-1 for stimulation of BMM DNA synthesis suggesting a possible role for protein kinase C in the stimulation of BMM DNA synthesis. In this report we show that several agents which raise intracellular cAMP (8-bromoadenosine 3':5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, cholera toxin, and prostaglandin E2) reversibly inhibit DNA synthesis in BMM induced by CSF-1, granulocyte macrophage-colony stimulating factor, interleukin-3, and PMA. The suppressive action of cAMP elevation on the proliferative response to CSF-1 can be manifested even late in the G1 phase of the cell cycle. Several CSF-1-stimulated earlier responses, viz. protein synthesis, Na+/H+ exchange, Na+,K(+)-ATPase and c-myc-mRNA expression, were not inhibited thus showing a striking difference from some other cellular systems involving growth factor-mediated responses. c-fos-mRNA levels were raised and stabilized by the cAMP-elevating agents, and this modulation was not altered by CSF-1. Thus, the signaling pathways in the macrophages involving tyrosine kinase and protein kinase C activation are associated with increased proliferation while those involving elevation of cAMP (and presumably activation of cAMP-dependent protein kinases) appear to have an inhibitory effect.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.