Adhesion, transendothelial migration, and reverse transmigration of in vitro cultured dendritic cells.

Dendritic cells (DC) are migratory cells which exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). The underlying mechanisms involved in these processes are still ill defined. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and ECM. DC were differentiated from monocytes by in vitro exposure to granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days. In adhesion assays a considerable proportion of DC bound to resting EC monolayers: (17% +/- 4%, mean +/- SE of eight experiments). Adhesion to tumor necrosis factor (TNF)-activated EC was increased to 29% +/- 5% (n = 8). Binding to resting EC was strongly inhibited by anti-CD11a and CD11b, but not by CD11c monoclonal antibodies (MoAbs); on TNF-activated EC, anti-VLA-4 in concert with anti-CD18 inhibited adhesion by more than 70%. Binding to a natural ECM, derived from cultured EC, or to purified fibronectin was high: 52% +/- 6% (n = 8) involved VLA-4 and VLA-5 integrins. In a transmigration assay, 10% +/- 2% (n = 6) of input cells were able to cross the EC monolayer. Unlike adhesion, transendothelial migration was significantly reduced by anti-CD31 MoAb. The amount of DC transmigrated through a monolayer of EC was increased twofold to threefold by a defined set of C-C chemokines including RANTES, MIP1alpha, MIP5, and, to a lesser extent, by MIP1beta and MCP-3. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC (13% +/- 4% of input cells seeded) was able to migrate across the endothelial basement membrane and, subsequently, across the endothelial barrier (reverse transmigration). The adhesion molecules and chemoattractants characterized herein are likely to underlie the complex trafficking of DC in vivo.

Human monocyte-derived and CD34+ cell-derived dendritic cells express functional receptors for platelet activating factor.

Dendritic cells (DC) are a heterogeneous population of specialized antigen presenting cells that exhibit complex trafficking properties. DC differentiated in vitro from both peripheral monocytes and CD34+ cells expressed mRNA for platelet activating factor (PAF) receptor. Expression of PAF receptor was increased by TNF alpha, a prototypic inflammatory cytokine that induces differentiation and in vivo mobilization of DC. PAF induced in vitro directional migration of DC obtained from both precursor cells through its specific receptor. Since DC are highly motile cells, protein chemoattractants as well as bioactive phospholipids are likely to contribute to tissue localization of DC, in vivo.

Interleukin-1 and tumor necrosis factor production in acute non-lymphoid leukemia.

We have investigated interleukin-1 (IL-1) and tumor necrosis factor (TNF) release in 20 patients with acute non-lymphoid leukemia (ANLL) after culture with bacterial lipopolysaccharide (LPS) or in the absence of deliberate stimulation. IL-1 and TNF were identified by appropriate bioassays inhibitable by specific antibodies. The capacity to produce IL-1 was expressed by most ANLL cases investigated irrespective of the FAB (French, American, British) subtype. However, the M4 and M5 cases tended to be better producers of IL-1 than M1-M3 cases. In contrast, TNF release was only restricted to M5 leukemias (3 out of 4 cases examined). Cytokine production may therefore provide additional criteria for a functional classification of ANLL. A considerable proportion of ANLL cases (7/18 bone marrow samples and 12/20 blood samples) released appreciable quantities of IL-1 in culture in the absence of deliberate stimulation. "Spontaneous" TNF production was also detected in 1 out of 3 M5 cases. Cells were cultured under LPS-negative conditions and polymixin B did not affect spontaneous cytokine release. Moreover, Northern blot analysis showed that freshly isolated, non-cultured ANLL cells expressed IL-1 beta transcripts. Inasmuch as IL-1 is responsible for hemopoietin-1 activity and IL-1 induces colony stimulating factor production in various cell types, the observation of IL-1 production in ANLL suggests that this mediator may be involved in regulatory amplifying circuits of leukemic cell proliferation.

Cytotoxic effector function of B lymphoblasts.

EBV-transformed B lymphoblastoid cell lines and clones were cytotoxic in vitro against Actinomycin D-pretreated WEHI 164 sarcoma cells, a system in which mononuclear phagocytes were previously identified as effectors. Similarly, unlike resting B cells, normal B lymphoblasts stimulated for 72 hr with Staphylococcus aureus Cowan I and purified according to the expression of the B cell surface marker B1, demonstrated appreciable cytolytic activity. B lymphoblastoid cells and derived supernatants were cytotoxic for Actinomycin D-pretreated WEHI 164 cells, and killing was inhibited by an anti-lymphotoxin but not an anti-tumor necrosis factor antiserum. Thus, B lymphoblasts have cytotoxic potential, mediated by lymphotoxin or a lymphotoxin-like soluble product.

Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes.

Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.

Involvement of tumour necrosis factor in monocyte-mediated rapid killing of actinomycin D-pretreated WEHI 164 sarcoma cells.

Human and murine monocyte-macrophages kill actinomycin D (ActD)-treated WEHI 164 sarcoma cells in a 6-hr 51Cr-release assay (drug-dependent cellular cytotoxicity, DDCC). In this study, we have investigated the cytotoxic activity of human recombinant tumour necrosis factor (hrTNF) against untreated and ActD-treated WEHI 164 sarcoma cells. Human recombinant TNF when added to the 6-hr 51Cr-release assay killed ActD-treated targets at doses ranging from 33 to 0.33 ng/ml, whereas untreated targets were resistant to lysis. The kinetics of lysis of ActD-treated targets was similar for hrTNF and blood monocytes. The protease inhibitors phenyl-methyl-sulphonyl-fluoride (PMSF) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced the DDCC activity of monocytes, monocyte supernatants and hrTNF. Killing of drug-sensitized target cells by monocyte supernatants was totally inhibited by a rabbit anti-TNF serum. These, as well as previous data on the physicochemical properties of the soluble cytotoxic factor released by monocytes, suggest that rapid monocyte-mediated killing of ActD-pretreated WEHI 164 sarcoma cells involves TNF or TNF-like molecules.

Dissociation between induction of ornithine decarboxylase and oxidative burst by phorbol esters in a macrophage cell line.

In order to investigate the correlation between stimulation of superoxide generation and induction of ornithine decarboxylase (ODC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) we have used the macrophage cell line J774.16 and a clone derived from this line that, by contrast with the parental line, is unable to generate superoxides in response to TPA. No difference was observed between the normal and the defective cells, with respect to ODC induction by TPA over a wide range of TPA concentrations (0.2-5.0 micrograms/ml). Similar results were obtained comparing resident and caseinate-elicited mouse peritoneal macrophages. Although resident macrophages did not generate superoxides in response to TPA, they did not differ from superoxide-generating, caseinate-elicited macrophages with respect to ODC induction. These data suggest a dissociation between the stimulation of the oxidative burst by TPA and a growth factor-like effect such as ODC induction.

Rapid killing of actinomycin D-treated tumour cells by mononuclear phagocytes: reactivity in mouse strains with defective classical tumour cytotoxicity.

In confirmation of previous data, macrophages from C3H/HeJ, C57BL/10ScCR and A/J mice, exposed in vivo to BCG or in vitro to lymphokines, had little tumoricidal activity, as assessed in a 48-hr [3H]thymidine release assay against TU5 tumour cells, compared to macrophages from C3H/HeN, C57BL/6 and (BALB/c X DBA/2)F1 mice. Macrophages from these mouse strains were examined for their capacity to kill actinomycin D-pretreated WEHI 164 sarcoma cells in a 6-hr 51chromium release assay (drug-dependent cellular cytotoxicity, DDCC). Peptone-elicited macrophages from C3H/HeN, C57BL/6, (BALB/c X DBA/2)F1, C57BL/10ScCR and A/J mice had high DDCC activity, whereas C3H/HeJ macrophages expressed little cytotoxicity against actinomycin D-pretreated WEHI 164 cells. In vivo exposure to BCG or inactivated streptococci caused a modest augmentation of the DDCC effector function of C3H/HeJ macrophages, but levels of reactivity remained 20-fold less than those of similarly treated normal mice. Thus, C57BL/10ScCR and A/J macrophages have defective classical direct cytotoxicity but mediate DDCC efficiently, whereas C3H/HeJ macrophages are defective in both effector functions.

Rapid killing of actinomycin D-treated tumor cells by mononuclear phagocytes: characterization of effector cells in mice.

Pretreatment with Actinomycin D (Act D, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by mouse resident or peptone-induced peritoneal exudate cells (PEC) in a 6-hr 51Cr release assay. Cytotoxicity was attributed to cells of the monocyte macrophage lineage on the basis of tissue distribution, separation by adherence on plastic and carbonyl iron, membrane antigens, and expression in mice with defective T cell- or NK cell-mediated immunity. Macrophages from four strains of mice (C3H/HeJ, A/J, P/J, C57B1/10 ScCR) previously shown to have defective "classical" nonspecific tumoricidal activity were examined for killing of Act-D-treated WEHI 164 cells. C3H/HeJ peritoneal macrophages had little or no DDCC, whereas cells from A/J, P/J, and C57B1/10 ScCR mice had normal levels of this reactivity. Tumor cells exposed to ActD were heterogenous in their susceptibility to killing by PEC, with five lines showing significant, though variable, lysis, whereas 12 tumor lines, normal fibroblasts, and lymphoblasts were not appreciably killed under the same conditions. Macrophage-mediated DDCC was also detectable in a colony assay. DDCC could explain how macrophages contribute to the antitumor activity of selected chemotherapeutic agents in murine tumor models.

Monocyte function in intravenous drug abusers with lymphadenopathy syndrome and in patients with acquired immunodeficiency syndrome: selective impairment of chemotaxis.

We have investigated monocyte function in 17 intravenous drug abusers with the clinical and laboratory features of lymphadenopathy syndrome (LAS). LAS patients had normal numbers of circulating monocytes. Monocytes from LAS patients were comparable to cells from normal donors in terms of phagocytosis of latex beads, interleukin-1 secretion, O2- release and killing of antibody-sensitized lymphoma cells or actinomycin D pretreated WEHI 164 cells. In contrast 13 out of 17 LAS subjects tested in this respect as well as six out of nine AIDS patients showed a marked defect of monocyte chemotaxis. Thus monocytes from patients with LAS or AIDS have a selective defect of monocyte chemotaxis.

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors.

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA-induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.

Effects of inactivated streptococci (OK-432) on macrophage functions in mice.

The effects of inactivated streptococci (OK-432) on murine macrophage functions were investigated. In vivo treatment of peritoneal macrophages with OK-432 augmented the direct cytotoxic activity against TU5 tumor cells in a 48 h tritiated thymidine release assay. OK-432 also stimulated the rapid (6 h, 51Cr release) macrophage-mediated killing of Actinomycin D-sensitized WEHI 164 sarcoma cells. Moreover, the expression of la antigens on peritoneal macrophages was found to be greatly enhanced after in vivo treatment with OK-432. The immunomodulatory effects of OK-432 on macrophages functions may contribute to the antitumor activity of inactivated streptococci.

Rapid killing of actinomycin D-treated tumor cells--cytotoxicity of cell-free monocyte supernatants.

Human monocytes kill Actinomycin D-treated WEHI 164 sarcoma cells in a 6 h 51Cr release assay (drug dependent cellular cytotoxicity, DDCC). In the present study we have investigated and characterized the human monocyte production of a cytotoxic factor which mediates DDCC. Cell-free supernatants obtained culturing monocytes for 4-5 h kill Actinomycin D-treated WEHI 164 cells but not untreated tumor cells. A series of antiproteases inhibits the cytotoxic activity of cell-free monocyte supernatants, whereas scavengers of reactive oxygen intermediates were ineffective. The lytic activity was destroyed treating supernatants at 100 degrees C for 5 min or by exposure to acid pH or to proteinase K, whereas it was unaffected by heating at 56 degrees C for 30 min. Upon gel filtration on Sephacryl S200, cytolytic activity eluted in the 33,000 molecular weight range.

Detection of human mononuclear cell subsets by complement fixing monoclonal antibodies: evaluation of manual procedures.

Manual procedures to evaluate mononuclear cells reacted with monoclonal antibodies were evaluated with respect to reproducibility, ease of execution, and cost. The immunofluorescent technique and the complement-mediated cytotoxicity test produced very similar results; however, the latter was clearly preferable in terms of cost, time-consumption, and ease of execution. Evidence is also presented that purification of lymphocytes using gradient diffusion techniques may induce an uneven selectivity within the relative percentages of T cell subsets collected for analysis.