Accessory cell function of cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes.

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection. The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemia leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE2, PGD2, PGF2 alpha, TXB2 and 6-keto-PGF1 alpha. In all types studied PGE2 and TXB2 were the major products formed. The identification of PGE2 and TXB2 was confirmed by GC/MS with multiple ion monitoring. The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a person's lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test-negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage-specific CD68(+) epithelioid macrophages and small round CD3(+) lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid-fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead-based assay using the Luminex 100 and were found to contain interleukin (IL)-6, IL-8, interferon-gamma and tumor necrosis factor-alpha, cytokines known to be involved in human granuloma formation, in quantities from two-fold to 7000-fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.     

The pharmacologic regulation of interleukin-1 production: the role of prostaglandins

The role of prostaglandins in the regulation of lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) production by murine C3H/HeN resident peritoneal macrophages was studied. IL-1 production was initially studied in the presence of piroxicam and indomethacin, both inhibitors of prostaglandin biosynthesis. IL-1 was assayed using the IL-1-dependent proliferative response of C3H/HeJ thymocytes. LPS stimulation resulted in 15 to 20 ng/ml of prostaglandin E2 (PGE2) produced in the first hour of culture. IL-1-containing supernatants from drug-treated macrophages at dilutions of up to 1:32 resulted in enhanced thymocyte proliferation compared to control, non-drug-treated cultures and contained less than 2 ng/ml of PGE2. Similar enhancement of proliferation could be obtained by incubating non-drug-treated supernatants with monoclonal anti-PGE2 but not anti-thromboxane B2 (TxB2) antibody. Further dilutions of the drug-treated supernatants gave thymocyte proliferation responses which were indistinguishable from control cultures and, correspondingly, had identical values for IL-1 production. The absence of an effect on IL-1 production was confirmed by quantitation of intracellular IL-1 alpha using goat anti-IL-1 alpha antibody and by quantitation of supernatant IL-1 receptor competition assay. Exogenous PGE2, in the concentration range produced in macrophage supernatants (10-20 ng/ml), directly inhibited IL-1-stimulated thymocyte proliferation. Finally, when macrophages were stimulated with LPS for 24 hr in the presence of added PGE2, thymocyte proliferation was inhibited at the lowest supernatant dilutions, but as the IL-1-containing supernatants were diluted out, the assay curves were indistinguishable from non-PGE2-treated control. Thus, in this system, PGE2 has no effect on IL-1 synthesis, but rather has a direct inhibitory effect on thymocyte proliferation. Nonsteroidal anti-inflammatory drugs are not stimulating IL-1 production but are, in fact, relieving inhibition of the thymocyte IL-1 assay caused by the presence of prostaglandins.     

Fibroblast stimulation in schistosomiasis. V. Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor

Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis. We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro. These cells possessed functional and phenotyptic characteristics of activated macrophages. In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro. The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation. By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd. Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5. Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation. Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay. In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro. We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis.     

Differential Proliferative Characteristics of Alveolar Fibroblasts in Interstitial Lung Diseases: Regulative Role of IL-1 and PGE(2)

Fibroblasts (Fb) from patients with sarcoidosis (SA) and hypersensitivity pneumonitis (HP) exhibited a lower proliferative capacity compared with Fb obtained from control (CO) and diffuse interstitial fibrosis patients (DIF). Proliferation of Fb from SA or lip patients was suppressed by autologous LPS-stimulated alveolar macrophages (AM) supernatants but not by those from CO patients. Similarly, alveolar macrophages (AM) derived supernatant, obtained from CO, did not suppress the proliferation of SA and HP Fb. AM from SA and HP patients secreted higher amounts of IL-1alpha and beta compared with controls and compared with Fb from SA and HP patients. Steady levels of IL-1alpha and betamRNA were expressed in unstimulated and stimulated cultures. Fb from SA and HP patients could be stimulated by LPS to secrete significantly higher levels of PGE(2) than those detected in supernatants from LPS stimulated Fb of DIF patients. Only the proliferation of Fb from SA and HP patients was sensitive to amounts of IL-1 equivalent to those detected in the lung of these diseases. As SA and HP are two diseases where irreversible deterioration occurs in only 20% of the patients, we hypothesize that mediators in the lung may modulate Fb proliferation. IL-1 of AM origin and PGE(2) of Fb origin secreted at high levels, may be candidates for this suppression because it was abrogated by anti IL-1beta and indomethacin.     

Fibroblast stimulation in schistosomiasis. IV. Isolated egg granulomas elaborate a fibroblast chemoattractant in vitro

Hepatic fibrosis complicates the chronic granulomatous inflammatory reaction to Schistosoma mansoni eggs, and is the major cause of morbidity and mortality in human schistosomiasis. We previously presented evidence that schistosomal egg granulomas secreted factors that can stimulate fibroblast proliferation and collagen synthesis in vitro. We now report that serum-free supernatants from cultures of hepatic egg granulomas isolated from S. mansoni-infected mice contained activity that stimulated the directional migration of human and guinea pig dermal fibroblasts in modified Boyden chambers. This fibroblast chemotactic activity was also detected in culture supernatants of granuloma adherent cells highly enriched for macrophages (95% latex-ingesting) but not in culture supernatants from resident peritoneal macrophages of uninfected or infected mice. This suggests that granuloma macrophages are a source of the chemotactic activity. The chemoattractant had the properties of large molecular weight (greater than 200,000 daltons; Sephadex G-200 gel filtration), pl approximately 4.5 (preparative flatbed isoelectrofocusing in granular matrix), heat stability (56 degrees C; 45 min), and trypsin sensitivity. Since preincubation of the partially purified granuloma and adherent-cell derived chemoattractants with rabbit anti-human fibronectin antibody abolished their chemotactic activity, it appears that the factor is antigenically similar to fibronectin. We propose that egg granuloma macrophages are activated in vivo to secrete a fibronectin-like molecule with activity that stimulates the directional migration of fibroblasts. This factor may therefore play a role in the local recruitment of fibroblasts and, in concert with other granuloma-derived factors, may play an important role in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis

PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl cAMP (100 microM) significantly enhanced IgE and IgG1 production and diminished IgM and IgG3 synthesis. We also show that PGE and cholera toxin elevate intracellular cAMP in B lymphocytes in a dose-dependent manner. In contrast, PGF2 alpha (10(-6) M) and the B subunit of cholera toxin (100 pg/ml) did not increase cAMP and did not regulate the isotype of Ig produced, reiterating the importance of cAMP in enhancing isotype differentiation. Although PGE is known to inhibit a number of immune responses, our data show that it is not always inhibitory. PGE may play a role in atopy in vivo where PGE-secreting cells such as macrophages, follicular dendritic cells, and fibroblasts can promote IgE synthesis. This research emphasizes the importance of PGE in regulation of the humoral immune response and adds a new stimulatory action to the repertoire of known PGE effects.     

PGE2 and PGF2alpha biosynthesis in stimulated and nonstimulated peritoneal preparations containing macrophages

The biosynthesis of PGE2 and PGF2alpha was measured in intact peritoneal exudate preparations obtained from C. parvum-treated and control C3H mice. Although both the control and stimulated preparations biosynthesized PGF2alpha and PGE2 from [1-14C] arachidonic acid, the stimulated preparations generated more of both prostaglandins than did nonstimulated preparations, probably as a result of increased synthesis within macrophages. Increased transformation of PGE2 into PGF2alpha by PGE2 9-ketoreductase was noted in stimulated preparations when compared to that in control cells. The data suggest that stimulated macrophages are capable of generating increased quantities of PGF2alpha and therefore might function as one source of this substance in resolving inflammatory reactions.     

Cell-type specificity of interleukins 1alpha and 1beta on prostaglandin and plasminogen activator production in bovine endometrial cells

Interleukin (IL)-1alpha is a potent stimulator of prostaglandin production in bovine endometrium, and IL-1 affects plasminogen activator (PA) activity in several types of cells. In this study, we determined the effects of IL-1alpha and IL-1beta on production of the prostaglandins PGF(2alpha) and PGE(2) and on PA activity in cultured bovine endometrial epithelial and stromal cells. We also determined the effects of PGE(2) and PGF(2alpha) on PA activity in these cells. Finally, we used RT-PCR to examine the expression of IL-1alpha, IL-1beta, and IL-1 receptor type 1 (IL-1R) mRNA in cultured bovine endometrial cells. This analysis revealed that IL-1alpha mRNA was present only in the stromal cells, whereas IL-1beta and IL-1R mRNAs were present in both cell types. When cultured cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.006 to 3 nM for 24h, IL-1alpha and IL-1beta were found to dose-dependently stimulate PGE(2) and PGF(2alpha) production in stromal cells (P<0.05) but not in epithelial cells. On the other hand, exposure to IL-1alpha and IL-1beta dose-dependently increased PA activity in the epithelial cells, whereas neither stimulated PA production in the stromal cells. When cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.06 to 3 nM for 24h, the two IL-1s differed in their effects on both PGE(2) and PGF(2alpha) production in stromal cells and had significantly differed in their effects on PA activity in epithelial cells. Exposure to PGE(2) and PGF(2alpha) did not affect PA activity in either stromal or epithelial cells (P>0.05). Taken together, these results suggest the possibility that both IL-1alpha and IL-1beta are produced by the stromal cells, that IL-1beta is produced by the epithelial cells, and that IL-1alpha is a far more potent stimulator than IL-1beta of prostaglandin and PA production in cultured bovine endometrial epithelial and stromal cells.     

Enhancement of macrophage IL-1 production by Leishmania major infection in vitro and its inhibition by IFN-gamma

Peritoneal cells from highly susceptible BALB/c mice were infected with Leishmania major and cultured for various times in vitro. The culture supernatants contained significant levels of IL-1 which were consistently higher than those in the cell cultures stimulated with an optimal concentration of LPS. This finding extends to a macrophage cell line, P388D1, and peritoneal exudate cells stimulated with starch in vivo. However, the level of IL-1 produced was significantly reduced when the cells were preincubated with a lymphokine preparation (supernatant of Con A-stimulated rat spleen cells). The level of IL-1 produced seems to be directly correlated with the degree of parasitization of the macrophages. A similar and dose-dependent reduction in IL-1 production by infected macrophages could also be obtained when the cells were preincubated with IFN-gamma. This finding is in direct contrast to that of visceral leishmaniasis in which peritoneal macrophages from BALB/c mice infected with Leishmania donovani not only fail to produce IL-1 but also lose the capacity to produce IL-1. This apparent discrepancy is discussed in terms of a possible difference in the induction of cell-mediated immunity between the two leishmanial diseases.     

Release of IL-1 from mononuclear phagocytes

IL-1 alpha and -beta are 31- and 34-kDa cytokines produced by stimulated monocytes, macrophages, and a variety of other cells. These proteins are thought to function primarily as intercellular mediators and can be detected in plasma and the supernatants of cultured cells; however, IL-1 alpha and -beta contain no identifiable signal peptides and are not secreted via the classical secretory pathway. To understand the mechanism of IL-1 release, we have analyzed IL-1 production by LPS-stimulated mononuclear cells. IL-1 was quantified by bioassay, immunoprecipitation, and ELISA. Of these techniques, only immunoprecipitation permitted the quantitative detection of intracellular pro-IL-1. Both the full-length pro-forms and proteolytically processed mature forms of IL-1 were detected in culture supernatants; however, for macrophages the released material represented less than 5% of the total IL-1 alpha and -beta synthesized. Freshly isolated human monocytes released a higher fraction of their total IL-1 (up to 22%): however, monocytes cultured in vitro for 24 h showed very little fractional release, similar to macrophages. Nonspecific release of intracellular contents was determined by measurement of release of lactate dehydrogenase activity and was found to parallel IL-1 release. In fact the higher release of IL-1 from freshly cultured human monocytes correlated also with an increase in the release of lactate dehydrogenase. We conclude that, in cultured LPS-stimulated monocytic cells, IL-1 is not released via a novel secretory pathway, but exits the cell via a nonspecific pathway, most likely as a consequence of cellular injury.     

Influence of morphology and malignancy of three new human melanoma cell lines on the cyclooxygenase pathway

Three newly established human melanoma cell lines (WU-BI, PN-JC, MJ-ZJ) of different morphology and different stage of malignancy were incubated with ionophore A23187 (2.5 to 40 microM) or arachidonic acid (AA, 6.25 to 100 microM). PGF2 alpha, 6-keto-PGF1 alpha, PGE2, TXB2 and 2,3-dinor-TXB2 from isolated cells and supernatants were measured by negative ion chemical ionization gas chromatography/mass spectrometry (GC/MS). PGE2 decreased in the fibroblastoid MJ-ZJ cells from 36.7 ng/mg cell protein about 70% (A23187) and about 20% (AA), respectively. However, in the cell supernatant PGE2 increased up to 295.4 +/- 66.5 ng/mg cell protein. Production of PGF2 alpha and PGE2 increased up to 5.7 +/- 1.2 ng/mg cell protein for polydendritic WU-BI cells and spindle shaped PN-JC cells. Up to 9.3 +/- 4.3 ng PGF2 alpha and 13.4 +/- 4.7 ng PGE2 was measured for WU-BI and PN-JC in the cell supernatants. All three melanoma cell lines completely lacked formation of 6-keto-PGF1 alpha, TXB2, and 2,3-dinor-TXB2.     

Synthetic pathways of gallbladder mucosal prostanoids: the role of cyclooxygenase-1 and 2.

Acute cholecystitis is associated with increased gallbladder prostanoid formation and the inflammatory changes and prostanoid increases can be inhibited by nonsteroidal anti-inflammatory agents. Recent information indicates that prostanoids are produced by two cyclooxygenase (COX) enzymes, COX-1 and COX-2. The purpose of this study was to determine the COX enzymatic pathway in gallbladder mucosal cells involved in the production of prostanoids stimulated by inflammatory agents. Human gallbladder mucosal cells were isolated from cholecystectomy specimens and maintained in cell culture and studied in comparison with cells from a well differentiated gallbladder mucosal carcinoma cell line. COX enzymes were evaluated by Western immunoblotting and prostanoids were measured by ELISA. Unstimulated and stimulated cells were exposed to specific COX-1 and COX-2 inhibitors. In both normal and transformed cells constitutive COX-1 was evident and in gallbladder cancer cells lysophosphatidyl choline (LPC) induced the formation of constitutive COX-1 enzyme. While not detected in unstimulated normal mucosal cells and cancer cells, COX-2 protein was induced by both lipopolysaccharide (LPS) and LPC. Unstimulated gallbladder mucosal cells and cancer cells produced prostaglandin E2 (PGE2) and prostacyclin (6-keto prostaglandin F1alpha, 6-keto PGF1alpha) continuously. In freshly isolated normal gallbladder mucosal cells, continuously produced 6 keto PGF1alpha was inhibited by both COX-1 and COX-2 inhibitors while PGE2 levels were not affected. Both LPS and LPC stimulated PGE2 and 6 keto PGF1alpha formation were blocked by COX-2 inhibitors in freshly isolated, normal human gallbladder mucosal cells and in the gallbladder cancer cells. The prostanoid response of gallbladder cells stimulated by proinflammatory agents is inhibited by COX-2 inhibitors suggesting that these agents may be effective in treating the pain and inflammation of gallbladder disease.     

Prostaglandin induced shape changes in fibroblasts grown in cell culture

PGE2 induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE2 at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hours. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE2 resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF2 alpha did not however induce similar changes. This difference between PGE2 and PGF2 alpha is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE2 may have the ability to induce shape changes in cells.     

Monoclonal antibodies to murine IL-1 alpha. Production, characterization, and inhibition of membrane-associated IL-1 activity

We report the production of hamster anti-murine IL-1 alpha mAb and analysis of their specificity and suitability for use in murine IL-1 immunologic and biologic assays. The mAb bound to murine (Mu) rIL-1 alpha 3 but not rMuIL-1 beta as assessed by both direct ELISA and immunoprecipitation. They also inhibited the biologic activity of MuIL-1 alpha but not the activity of rMuIL-1 beta as measured in a T cell co-stimulator assay. These IL-1 alpha specific mAb only partially inhibited the co-stimulator activity found in macrophage culture supernatants but completely inhibited the co-stimulator activity of fixed peritoneal exudate cells. The data indicate that the species of IL-1 associated with murine macrophage membranes shares at least two epitopes with IL-1 alpha and probably represents a product of the IL-1 alpha gene. These reagents will be valuable for quantitative assessment of specific IL-1 proteins on cell surfaces, in culture supernatants, and in cell lysates. They will also be useful both in vitro and in vivo for determining the relative roles of the different IL-1 species in the development of biologic responses.     

Mononuclear cells enhance prostaglandin E2 production of polymorphonuclear leukocytes via tumor necrosis factor alpha

To clarify the interactions between mononuclear cells and polymorphonuclear leukocytes, and to identify the cytokine(s) that mediate the interaction, the effects of a culture supernatant of LPS-stimulated mononuclear cells on production of arachidonic acid metabolites of polymorphonuclear cells were studied. The culture supernatant of LPS-stimulated mononuclear cells increased production of prostaglandin E2 of polymorphonuclear cells. TNF alpha, but not IL-1, IL-2, IL-6, or IFN gamma, enhanced the prostaglandin E2 production when added in vitro. Additionally, an anti-rTNF alpha monoclonal antibody inhibited the stimulating activity of the culture supernatants. TNF alpha, produced by mononuclear cells, appears to play an important role in the development of inflammation, such as rheumatoid arthritis, by enhancing the arachidonic acid metabolism of the polymorphonuclear cells.     

Induction of tumoricidal activated macrophages by a liposome-encapsulated glycolipid, trehalose 2,3,6'-trimycolate from Gordona aurantiaca

A mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate, prepared from a non-pathogenic acid-fast bacterium Gordona aurantiaca, was shown to induce strong tumoricidal activity in peritoneal exudate cells by intravenous or intraperitoneal injection of liposome-encapsulated preparations. The mycolic acid derivative containing a high proportion of unsaturated fatty acids rendered macrophages cytotoxic against syngeneic mastocytoma cells in the absence of endotoxin, for over 14 days after the injection. The macrophages were ascertained to be at low intracellular levels of a lysosomal enzyme beta-galactosidase and an ectoenzyme alkaline phosphodiesterase, a specific pattern as previously described for "primed macrophages". However the culture supernatants of the peritoneal exudate cells were not cytotoxic.     

Regulation of macrophage-mediated tumor cell killing by prostaglandins: comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), the latter detected as its stable metabolite, 6-keto PGF1 alpha. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10(-7)M for PGI2 and 3 x 10(-8)M for PGE2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE2 and PGI2; however, only PGE2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.