Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Purification and biochemical characterization of a human autocrine growth factor produced by Epstein-Barr virus-transformed B cells

Autocrine growth factor for Epstein-Barr virus-transformed human B cells (aBGF), a protein that is constitutively produced by the human EBV-transformed B cell line 5/2, has been purified from serum-free conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The purified protein has a m.w. of 16,000 in NaDodSO4/polyacrylamide gel electrophoresis and an isoelectric point between 7.0 and 8.0. The relative molecular mass 16,000 form exists in equilibrium with dimeric and tetrameric forms. aBGF supports the growth of EBV-transformed B cells, which have been deprived of their own conditioned medium. The purified aBGF is fully effective at 0.5 ng/ml and has no interleukin 1 activity in the lymphocyte activation factor assay. Because several randomly selected lines of EBV-transformed cells and one EBV-negative lymphoma cell line both produce aBGF activity and show growth dependency on aBGF and because stimulation of normal B cells with anti-immunoglobulin M is increased by aBGF, we propose that aBGF has general significance for growth control of human B cells.     

Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide

CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.     

Isolation and characterization of a neu protein-specific activating factor from human ATL-2 cell conditioned medium.

The rat neu gene product is a 185 kD membrane bound tyrosine kinase that is closely related to, yet distinct from the epidermal growth factor receptor. The biochemical and cellular effects of a neu protein-specific activating factor (NAF) detected in human ATL-2 cell conditioned medium were recently described (1). To further characterize NAF, some of its physicochemical properties were examined and a method for purifying this factor from ATL-2 cell conditioned medium was developed. In these studies NAF was found to be heat stable and sensitive to the protease chymotrypsin. In addition, a method for purifying this activity was developed using a quantifiable, in vitro autophosphorylation assay system to measure NAF activity in fractions following ion-exchange and then reverse-phase HPLC.     

Physicochemical and functional properties of murine B cell-derived B cell growth factor II (WEHI-231-BCGF-II)

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture

A protein chemotactic for peripheral blood monocytes (SMC-CF) of potential importance in their recruitment to the arterial intima in atherogenesis was purified from serum-free medium conditioned by cultured baboon aortic medial smooth muscle cells. The purification of SMC-CF was monitored by a filter assay using human peripheral blood mononuclear cells and was achieved by batch separation on a cation-exchange gel followed by gel permeation chromatography, ion-exchange high-performance liquid chromatography (HPLC), and reversed-phase HPLC. The overall recovery was approximately 10% of the initial activity and yielded 0.5-1 microgram of SMC-CF/L of conditioned medium. On analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SMC-CF migrated as a monomeric protein with an apparent molecular weight of 14,500. A dose-dependent relationship was observed between SMC-CF concentration and monocyte chemotactic activity, with maximal and half-maximal biologic activity being observed at approximately 5 and 0.1 nM, respectively. Cultured baboon aortic smooth muscle cells also express the genes for both the A and B polypeptide chains of platelet-derived growth factor, which has been reported to be chemotactic for blood monocytes and neutrophils [Deuel, T. F., Senior, R. M., Huang, J. S., & Griffin, G. L. (1982) J. Clin. Invest. 69, 1046-1049]. Amino acid composition analyses indicate that SMC-CF is not derived either from polypeptide chain of this growth factor or from certain potentially chemotactic connective tissue proteins.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.     

Constitutive production by the WEHI-3 cell line of B cell growth and differentiation factor that co-purifies with interleukin 1

The myelomonocytic cell line WEHI-3 produces constitutively a factor that affects the growth and differentiation of murine B cells in culture. This cell line also secretes colony-stimulating factors (CSF), interleukin 1 (IL 1) but not interleukin 2. Sequential purification through AcA54 gel filtration, DEAE-Sephacel ion exchange chromatography, and buffer electrofocussing clearly resolved the B cell growth and differentiation factor (BGDF) from the CSF activities but failed to separate BGDF from IL 1. The WEHI-3-derived material responsible for BGDF/IL 1 activity, however, exhibited different behavior on DEAE chromatography (elution at 175 mM NaCl) to that reported for IL 1 from the P388D1 cell line (elution at 50 mM NaCl). B cell growth and differentiation could be induced by WEHI-3 BGDF/IL 1 in cultures of normal spleen cells depleted of T cells and adherent cells but not in cultures of spleen cells from B cell-deficient CBA/N mice, even though thymocytes from such mice displayed a normal response to IL 1. Significant B cell proliferation induced by BGDF/IL 1 was apparent only in the presence of submitogenic concentrations of anti-mouse IgM antibodies, but under these conditions few antibody-forming cells (AFC) were generated. In contrast, B cell differentiation to AFC occurred in the presence of the factor alone, and this response was inhibited by anti-IgM. Thus there appeared to be a reciprocal relationship between B cell proliferation and differentiation induced by BGDF/IL 1. The significance of these results is discussed in the light of other recent studies of BGDF.     

Functional properties of two human B cell growth factor species separated by lectin affinity column

The supernatants from PHA-activated normal human T cells (conditioned medium) were fractionated by differential adsorption on Con A-Sepharose columns. The effluent contained both IL 2 activity (tested on the CTL-L2 cell line) and BCGF activity (tested on anti-mu-activated normal B-enriched cells). Absorption of such effluent on PHA-activated T cell blasts removed the IL 2 activity without affecting the BCGF activity. The eluate also displayed BCGF activity (tested on anti-mu-activated B-enriched cells) without detectable IL 2 activity. The two BCGF species isolated by this criteria synergize with semipurified IL 1 to support the anti-mu-induced proliferation of highly monocyte-depleted B cells. Both BCGF species required the presence of anti-mu Ab to support the proliferation of small B cells (at the G0 stage). However, in contrast to crude conditioned medium and to the BCGF present in the effluent, the BCGF present in the eluate was unable to induce the proliferation of unfractionated or large (at the G1 stage) B cells in the absence of anti-mu Ab. These results support the existence of two functionally different human BCGF species, differing at least in their relative sugar content and distinct from IL 2.     

Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species.

Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of 20-25 pM was detected at 40-720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.     

Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Induction of interleukin 2 from a murine B cell tumor by a factor found in immune serum

The murine B cell tumor line 2 PK-3 secretes T cell growth factor activity after incubation for 6 to 48 hr with a factor present in heterologous immune serum. T cell growth factor derived from 2 PK-3 was compared with IL 2 produced by the Con A-induced T lymphoma cell line EL-4 G12. These studies indicated that T cell growth factor activities derived from both cell lines were similar with respect to m.w., pI values, and the ability to support growth of two IL 2-dependent T cell clones. Three preparations of immune sera were found to be active in the induction of IL 2 activity from 2 PK-3 cells, including rabbit anti-mouse brain, rabbit anti-complete Freund's adjuvant, and goat anti-mouse Ig. None of these preparations, however, induced IL 2 from EL-4 G12 cells. It was also observed that LPS synergized with immune serum to produce enhanced activity. Normal sera prepared from unimmunized animals were not active in the induction of IL 2 activity. Fractionation of immune serum on protein A Sepharose suggested that the IL 2-inducing agent is not IgG.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.     

Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan

Mouse mast cells were differentiated and grown by culturing bone marrow cells in medium containing 2 X 10(-10) M purified interleukin 3 (IL 3). The cells obtained were similar in ultrastructure, membrane antigen phenotype, proteoglycan type, and lipid products generated upon immunologic activation to mast cells differentiated in culture by WEHI-3-conditioned medium (WEHI-3-CM) and by concanavalin A (Con A) splenocyte-conditioned medium. Phenotypically, these cells expressed IgE receptors and H-2 antigens and were recognized by a monoclonal antibody (B23.1) that did not react with mouse serosal heparin-containing mast cells. The classic phenotypic markers of mouse T cells or macrophages were not detected. The mouse mast cells differentiated with IL 3 as well as those differentiated in WEHI-3-CM incorporated [35S]sulfate into a nonheparin proteoglycan of 150,000 to 200,000 m.w. Most of the 35S-labeled macromolecules were degraded by chondroitinase ABC to yield only two disaccharides, which co-chromatographed on ascending thin layer chromatography with delta Di-4S and delta Di-diSE; thus, the proteoglycan in these cells is composed of chondroitin sulfate E glycosaminoglycans. After sensitization with monoclonal IgE, washing, and antigen activation, the IL 3 differentiated cells released the preformed mediator beta-hexosaminidase and generated and released two major classes of lipid mediators. The quantities of leukotriene C4 (LTC4), leukotriene B4 (LTB4), and platelet-activating factor (PAF-acether) generated/10(6) cells were 17, 3.0, and 3.1 ng, respectively. The ratio of these three lipid mediators was similar to that obtained from mast cells differentiated in WEHI-3-CM and in Con A-conditioned medium. Thus, T cell-derived IL 3 is the component present in the conditioned media that is required for differentiation and growth of the subclass of mast cells containing chondroitin sulfate E proteoglycan, designated E-MC. The IL 3-dependent E-MC may represent the in vitro counterpart of the T-cell-dependent mucosal mast cell, suggesting in turn that the production of LTC4 and LTB4 and of PAF-acether may play a role in adaptive intestinal immunity to helminthic parasites.