IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor.

We have identified and purified a factor that inhibits the production of IL-1 beta and TNF by stimulated human mononuclear cells. The activity is produced by the T cell lines Hut-78 and Mo constitutively under serum-free conditions. Crude conditioned media have titers of up to 100 U/ml (one unit defined as the reciprocal of the dilution producing 50% inhibition). The activity resides mainly in a single size peak of 30 to 35 kDa and an isoelectric point around 8. Other cytokines in this size range that have been reported to be inhibitory for IL-1 and TNF production include TGF-beta, IL-4, and IL-6; these factors were excluded by lack of detection, neutralizing antibody, and low activity compared with our factor. Another factor with these size and charge properties is IL-10, which inhibits T cell cytokine production. By polymerase chain reaction analysis, Mo and HuT-78 lines contain IL-10 transcripts whereas JURKAT is negative; this correlates with inhibitor bioactivity from the three lines. Use of mAb specifically showed the inhibitor to be IL-10.

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor.

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.

Stimulation of macrophage antibody-dependent killing of tumor targets by recombinant lymphokine factors and M-CSF.

Macrophage colony-stimulating factor (M-CSF) was investigated as a stimulator of ADCC to the murine R1.1 thymoma target by murine peritoneal exudate macrophages which were elicited by proteose peptone. Both an 125IUdR release and a viable cell count assay were used. The latter assay avoids radiation damage, and the fate of the targets can be determined over a long period. Pretreatment of macrophages for several days in culture with lymphokine (LK) from concanavalin A-induced mouse spleen cells moderately stimulated ADCC. Preincubation of macrophages with conventional or recombinant human M-CSF or immunoaffinity-purified mouse M-CSF alone had little effect. However, M-CSF greatly enhanced ADCC to the tumor target when used as a costimulant with LK, IFN-gamma, IFN-alpha, IFN-beta, or IL-2 to pretreat macrophages. Incubation of macrophages with LK or LK plus M-CSF for 2 days generated stronger ADCC than 1- or 3-day incubations. Enhancement of LK-stimulated ADCC by M-CSF appeared to plateau at about 1000 U/ml. The enhancement of macrophage cytotoxicity when stimulated with IFNs or IL-2 was most effective at the lowest active concentration of these LKs. At 1 U/ml IFN-gamma or IL-2, or 5 U/ml IFN-alpha or IFN-beta, M-CSF boosted ADCC activity to that using 10-fold of the LK alone. IL-1, IL-4, and TNF had little or no stimulating activity for ADCC alone or with M-CSF, and the other hemopoietic growth factors IL-3 and GM-CSF did not promote this effector function alone or with IFN-gamma. We previously showed that M-CSF boosted macrophage antibody-independent killing of TU5 sarcoma targets with or without LK (Cell. Immunol. 105, 270, 1987). These studies thus show that M-CSF is a positive regulator of both macrophage-nonspecific tumor lysis and ADCC.

Role of interleukin 2, interleukin 4, and alpha, beta, and gamma interferon in stimulating macrophage antibody-dependent tumoricidal activity.

Pretreatment of murine peritoneal exudate macrophages with 1-5 U/ml rIFN-gamma or rIL-2, or higher concentrations of IFN-alpha or IFN-beta greatly stimulated ADCC to Rl lymphoma targets. The assay was direct counting of viable target cells after 9 and 24 h using an E/T ratio of 5:1. 2d of pretreatment was optimal for enhancing ADCC. rIL-4 was inactive and IL-4-depleted Con A-induced spleen lymphokine retained its ADCC-stimulating activity. Antibody to IFN-gamma blocked the ADCC-promoting effect of the lymphokine, suggesting a major role for this factor.

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.

Independent regulation of B-cell inducing factor and IL-2 production by T lymphocytes, and direct and indirect promotion of immunoglobulin secretion by glucocorticosteroid.

The conditions for induction of B-cell inducing factor (BIF) by human peripheral blood T cells was investigated. BIF was assayed by induction of immunoglobulin secreting cells (ISC) in peripheral blood B (non-T) cells stimulated with Staphylococcus aureus bacteria strain Cowan I (Sac), and in the IgM cell line SKW6.4. Maximum BIF production occurred with high concentrations of the T-cell mitogens phytohemagglutinin, concanavalin A, and PWM. Dexamethasone (Dex) also induced BIF production in T cells at 10(-5) to 10(-7) M. At 10(-5) and 10(-6) M Dex, the T-cell supernatants had to be dialyzed before testing because Dex alone stimulated variable levels of ISC in both test B-cell assays. Dex did not enhance BIF production by T cells that were optimally stimulated by lectin. BIF levels were maximum by Day 2 of T-cell cultures and remained high at Days 3 and 4. In contrast, IL-2 reached a peak at Day 1 and declined drastically by Day 4. We previously showed that IL-2 at less than 100 U/ml did not induce ISC in B cells and did not alter ISC induction by BIF. Dex did not induce IL-2 production and inhibited IL-2 production induced by Con A, in contrast to the promoting effects of Dex on BIF production, providing further evidence for the independence of BIF and IL-2 production and B-cell stimulation.

Biological properties and molecular biology of the human macrophage growth factor, CSF-1.

CSF-1 is a growth and differentiation factor for the production of mononuclear phagocytes from undifferentiated bone marrow progenitors. In addition to previously described effects on mature cells, we show here that CSF-1 stimulates the production by monocytes of interferon, tumor necrosis factor, and myeloid CSF that produces mainly mixed neutrophil-macrophage colonies in bone marrow culture. Pretreatment with CSF-1 also promotes resistance to viral infection and tumor cytotoxicity in murine peritoneal macrophages. Based on amino acid sequence data of purified human urinary and murine L cell CSF-1, we have cloned the complementary DNA (cDNA) from messenger RNA (mRNA) of the human CSF-1 producing MIA PaCa cell line. The cDNA specifies a 32 amino acid signal peptide followed by a protein of 224 amino acids. Several facts suggest, however, that one-third of the molecule at the C-terminal end is processed off intracellularly to derive the secreted growth factor. The gene is about 18 kilobases (kb) in length and contains 9 exons. Although there appears to be a single copy gene for CSF-1, cells expressing the factor contain several mRNA species, suggesting that the gene may have several functions or levels of regulation. High level expression of the recombinant protein will allow preclinical testing in several disease models for therapeutic efficacy that has been suggested from in vitro and in vivo biological properties of CSF-1.

Rescue of IgM, IgG, and IgA production in common varied immunodeficiency by T cell-independent stimulation with Epstein-Barr virus.

We previously defined three categories of B-cell defects in common varied immunodeficiency (CVI): failure to produce IgG and IgA in response to T cell-dependent (TD) stimulation by Staphylococcus bacteria (Sac) plus pokeweed mitogen or B-cell inducing factor (BIF), failure to produce any immunoglobulin, and failure of Sac-induced proliferation and differentiation. The present study includes the responses of 22 CVI patients to T cell-independent (TI) stimulation by Epstein-Barr virus (EBV). In the majority of patients, EBV-stimulated B cells showed normal proliferation and IgM production. In addition, IgG and IgA production was in the range of that for EBV-stimulated normal cells in many patients. Among 11 patients with no TD production of immunoglobulin of any isotype, two showed normal IgM secretion in response to EBV and five others had significant but subnormal responses. Four patients never had humoral responses despite repeated testing and removal of potentially suppressing T cells and monocytes. Concanavalin A stimulation of the T cells from all the patients tested resulted in the production of B-cell inducing factor at higher levels than for normal donor T cells, as assayed on normal Sac-stimulated B cells. These results show that many cases of B-cell defects in CVI patients involving TD production of IgM, switching to TD production of IgG and IgA, and mitogen responses to Sac are not absolute defects. The B cells will respond normally to some stimuli.

Human B cell-inducing factor(s) for production of IgM, IgG and IgA: independence from IL 2.

Human peripheral blood B cells are stimulated into proliferation by killed Staphylococcus aureus bacteria strain Cowan I (Sac). T lymphocytes in the presence of a T cell mitogen induce high numbers of immunoglobulin-secreting cells (ISC) in these Sac-stimulated B cells. The T cells can be largely replaced by a lymphokine factor. We describe here the 11000-fold purification of this B cell-inducing factor (BIF). BIF preparations that are free of IL 2 do not require IL 2 for optimal induction of ISC. This was shown by the lack of effect of IL 2 alone or with suboptimal or optimal concentrations of BIF on the induction of ISC and by the absence of IL 2 production in the purified B cell population which, with other controls, excludes significant T cell contamination. BIF, purified through four fractionation steps and free of IL 2, induces IgM, IgG, and IgA-ISC in approximately the same ratio as unfractionated lymphokine. Because we have not yet attained a pure BIF preparation, the possibility of separate factors for the production of each immunoglobulin isotype cannot be ruled out.

Cooperation of IgG monoclonal antibodies in macrophage antibody-dependent cellular cytotoxicity (ADCC) to tumor targets.

Six IgG monoclonal antibodies representing the four murine IgG isotypes were active individually in ADCC to T-lymphoma targets mediated by murine macrophages and human blood K cells. The monoclonals were directed against four antigens (Thy-1.2, H-2k, Ly 2.1, Ly 9.2). None was as effective in ADCC as allo-anti-Thy-1.2 serum or rabbit anti-mouse spleen serum, even at plateau levels of killing. Monoclonals gave the highest levels of ADCC at relatively low amounts of antibody bound to targets; increasing the amount of bound antibody by 10- to 100-fold did not increase murine macrophage-mediated cytotoxicity. In contrast, ADCC using alloantiserum continued to increase over the same range of antibody bound to the tumor targets. The activity of individual monoclonals was not enhanced by the presence of various dilutions of normal mouse or rabbit serum, suggesting that the superiority of the allo- and hetero-antisera was due to their content of heterogeneous antibodies. IgG monoclonals of different isotypes and recognizing different antigens gave enhanced ADCC in combination; monoclonals to the same antigen did not. An IgM anti-Thy-1.2 monoclonal was inactive in ADCC and inhibited the activity of IgG monoclonals of the same specificity. These studies show that IgG antibodies of different specificity and class can collaborate in ADCC, and that this cooperative effect is not due simply to increased amounts of antibody bound to the tumor targets.

Cell-mediated lysis of tumor targets directed by murine monoclonal antibodies of IgM and all IgG isotypes.

Seven murine monoclonal antibodies to antigens expressed on T lymphoma targets were tested for directing antibody-dependent cellular cytotoxicity (ADCC). Peptone-induced peritoneal exudate macrophages, the LPS-stimulated RAW264.10 cell line, and human blood nonadherent mononuclear leukocytes were used as effector cells. All six IgG monoclonals tested, representing the four murine IgG isotypes and directed against four antigens (Thy-1.2, H-2k, Ly-2.1, Ly-9.2), were all active in ADCC. In contrast, an IgM anti-Thy-1.2 showed no activity despite very high C-cytotoxic titers. Thus, there does not seem to be any restriction among IgG classes for directing ADCC to tumor targets mediated by murine macrophages or human K cells.

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities.

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.

Differences in antibody-dependent cellular cytotoxicity and activated killing of tumor cells by macrophage cell lines.

A series of murine macrophage cell lines was assayed for killing of B and T lymphocytic and myeloid tumor targets by radiolabel release at effector:target ratios of 20:1. One group of lines was inactive in all assays. Another group of lines showed moderate spontaneous cytotoxicity to lymphoid tumors that was greatly enhanced by inclusion of antibody, lipopolysaccharide, or phorbol myristate acetate in the 22-hr assays. Addition of lymphokine to the assays induced only moderate killing. Pretreatment of cell lines with lipopolysaccharide, phorbol myristate acetate, lymphokine, or nonlymphocyte sources of macrophage colony-stimulating factors did not activate "angry" or nonspecific killing. However, such pretreatment greatly stimulated antibody-dependent cellular cytotoxicity. Cytotoxicity was not due to crowding or poor culture conditions: macrophage line supernatants were not toxic; rapidly growing lymphoid lines used in place of macrophages did not kill; and high macrophage concentrations (10(6)/ml) had reduced cytotoxic activity. These experiments show that the same cell type can mediate both activated killing and antibody-dependent cellular cytotoxicity of tumor targets. The active macrophage lines appear to be qualitatively similar to each other and to peritoneal exudate populations in tumoricidal activity. We showed previously that these macrophage lines could also phagocytose and lyse antibody-coated red blood cells. The pretreatment experiments suggest that the antibody-dependent cellular cytotoxicity state can be regulated independently of macrophage nonspecific tumoricidal capacity. These culture line models of macrophages offer several advantages in the analysis of cytotoxicity.

Cell interactions influencing murine marrow megakaryocytes: nature of the potentiator cell in bone marrow.

Auxiliary bone marrow cells are required for optimal murine megakaryocyte colony formation in addition to progenitor cells and a colony stimulating activity (CSA) present in WEHI-3 cell conditioned medium. These auxiliary cells are adherent, with a sedimentation rate of 5.8 mm hr-1 and buoyant density of 1.065-1.078 gcm-3. The activity from bone marrow cells is loss at irradiation doses above 900 rad. Bone marrow cells with these characteristics, and supernatants from lung, bone shafts, and peritoneal exudate cells were all active in enhancing megakaryocyte colony incidences in mouse bone marrow cultures above those stimulated by an obligatory activity in WEHI-3 cell conditioned medium. Certain macrophage cell lines (J774, P388D1) could elaborate the activity. This study confirms that a potentiation activity enhances CSA stimulation of megakaryocyte colony formation. The potentiator is elaborated by bone marrow cells in limiting amounts requiring either high cell concentrations or an exogenous source of the activity for optimal colony growth.

All classes of murine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes.

Antibody-dependent phagocytosis and lysis of sheep RBC by mouse peritoneal exudate cells were studied using IgG subclass fractions of sera prepared by protein A-Sepharose chromatography. Three IgG1, 3 IgG2a, 4 IgG2b, and 1 IgG3 hybridoma antibodies to RBC were also used. The results showed that some monoclonal IgG2a and IgG2b antibodies were more active than IgG1 or IgG3 preparations when normalized to hemagglutinating activity, but all IgG sources mediated both effector mechanisms. This is in accord with findings of strong Fc receptors on macrophages for the IgG2 isotypes and weaker binding by IgG1 and newly discovered IgG3 receptors. A monoclonal IgG2b with no hemagglutination titer showed normal cooperation with macrophages. In contrast, 2 hybridoma IgM were inactive in macrophage-mediated lysis and allowed only marginal ingestion. Similar results were obtained using several macrophage cell lines, mouse spleen and human peripheral blood mononuclear cells, showing that all these types of effector cells do not distinguish among (mouse) IgG subclasses in handling antibody-coated RBC.