Activation of human monocytes by LPS and DHEA.

Dehydroepiandrosterone (DHEA) alone, whatever the concentration used, or lipopolysaccharide (LPS) alone at 0.2 ng/ml did not induce the release of interleukin-6 (IL-6) or tumor necrosis factor (TNF) by human monocytes. However DHEA (10[-9] M or 10[-12] M) in association with LPS (0.2 ng/ml) did induce the release of IL-6 and TNF. When human monocytes were activated by 1 microg/ml LPS, both IL-6 and TNF secretions were observed. Monocytes activated by both DHEA (10[-9] M or 1O[-12] M) and LPS (1 microg/ml) secreted IL-6 and TNF at a higher level than that observed for monocytes activated only by LPS (1 microg/ml) alone. DHEA alone, whatever the concentration used, or LPS alone at 0.2 ng/ml did not induce the activation of mitogen-activated protein kinases (MAPkinases) and protein kinase C (PKC) or the expression of c-fos and c-jun. However DHEA (10[-9] M or 10[-12] M) and 0.2 ng/ml LPS together induced the activation of both MAPKinases and PKC and the expression of c-fos and c-jun. Furthermore, the activation of PKC and MAPKinases and the expression of c-fos and c-jun were much greater when human monocytes were activated by both LPS (1 microg/ml) and DHEA (10[-9] M or 10[-12] M) than when the monocytes were activated only by LPS at 1 microg/ml. Therefore, DHEA and LPS displayed a synergistic effect on monocyte activation.

Signal transduction in LPS-activated aged and young monocytes.

Aged monocytes, that is, monocytes purified from the blood of donors > or =65 years of age, when compared with young monocytes, that is, monocytes purified from the blood of young donors 25 years of age, display a decrease in interleukin-6 (IL-6) and tumor necrosis factor (TNF) production after activation by lipopolysaccharide (LPS). The LPS concentration required to obtain IL-6 and TNF production is much higher for aged monocytes than for young monocytes. Furthermore, the intensity of TNF and IL-6 production was much weaker for LPS-activated aged monocytes than for LPS-activated young monocytes. In addition, deficient protein kinase C (PKC)-alpha, PKC-/betaI, and PKC-betaII activation, deficient mitogen-activated protein kinase (MAP-Kinase) activation, and deficient expression of c-Fos and c-Jun was observed in LPS-activated aged monocytes when compared with LPS-activated young monocytes. These data suggest that age induces human monocyte immune deficiencies that could be observed not only at the functional level but also in the signal transduction pathways.

Localization of an entrapped item within unilamellar vesicle compartments: use of ultrasound disruption as a procedure to separate aqueous phase and lipidic lamellae.

A procedure has been carried out previously to separate the aqueous phase and the lipidic lamellae from multilamellar liposomes (Bakouche and Gerlier, Analyt. Biochem., 1983, 130, 379). This procedure consisted in multiple short bursts of sonication at a temperature below the transition temperature (Tm) of the lowest melting component followed by an ultracentrifugation. The aqueous phase and the lipidic lamellae were recovered in the supernatant and in the pellet respectively. This procedure was adapted for unilamellar vesicles by modifying the second step of the procedure. A suspension of unilamellar liposomes containing 5,6-carboxyfluorescein (5,6-CF) as a probe for the aqueous phase was disrupted by sonication at low temperature. After gel filtration on to a Sephadex G100 column, the phospholipid bilayers were readily separated from the aqueous probe 5,6-CF. Such a procedure should allow the localization of any item within the unilamellar liposome compartments.

Physical separation of the aqueous phase and lipoidal lamellae from multilamellar liposomes: an analytical and preparative procedure.

An efficient and rapid method to separate the aqueous phase from lipoidal lamellae after mechanical disruption of multilamellar liposomes with preservation of the phospholipid bilayer organization has been designed. Liposome suspensions were subjected to a few short bursts of sonication at a temperature below the transition temperature (Tc) of the lowest melting phospholipid component. This was followed by ultracentrifugation. The aqueous supernatant contained more than 90% of the encapsulated aqueous elements (6-carboxyfluorescein, bovine serum albumin) and less than 1% of the lipids. The washed pellet consisted of lipoidal lamellae devoid of any encapsulated aqueous phase. The lipoidal lamellae contained more than 99% phospholipids and were contaminated with less than 0.1% aqueous components. Such a method should allow the localization of any component within the aqueous phase or lipoidal lamellae of the liposomes.

Enhancement of immunogenicity of tumour virus antigen by liposomes: the effect of lipid composition.

The secondary humoral response evoked in W/Fu rats by the weakly immunogenic soluble Gross cell surface antigen (GCSAa) extracted from the syngeneic (C58NT)D lymphoma can be enhanced when GCSAa is presented in liposomes, and this requires the antigen to be strongly associated with the phospholipid bilayers. In order to investigate further the role of the phospholipid microenvironment in the membrane presentation of this antigen, the relationship between the phospholipid composition and the immunogenic potency of GCSAa liposomes was explored. For a given neutral phospholipid component, optimal immunogenicity was obtained when 20% cholesterol was present and when a negatively charged phospholipid was included as a minor component. When phosphatidylcholines (PC) were used as the major neutral component, the immunogenicity of GCSAa liposomes from optimal for distearoyl PC (DSPC) decreased when decreasing the PC acyl chain length down to the background level for dilauroyl PC (DLPC). Similarly, the use of PC with acyl chain of increasing unsaturation was followed by a decline in the immunogenicity of the GCSAa liposomes up to the background level for dilinoleoyl PC (DLiPC). Replacing PC headgroups by phosphatidylethanolamine (PE) headgroups abolished the enhancing effect of the liposome presentation on GCSAa immunogenicity. Three groups of phospholipids unable to promote the expression of the GCSAa immunogenicity could be distinguished: the DLPC, DLiPC group, unable to prime the animals but, contrary to the soluble antigen, able to boost the animal after an appropriate priming with GCSAa-DSPC-liposomes, the dimyristoyl PE group, unable to prime or to boost and non-toxic in vitro for the macrophages, and the dipalmitoyl PE group, acutely toxic for macrophages.

Presentation of an MuLV-related tumour antigen in liposomes as a potent tertiary immunogen after adoptive transfer.

The immunogenicity of Gross virus cell surface antigen (GCSAa) extracted from rat lymphoma cells can be dramatically increased by its presentation into liposomes, probably by mimicking the cell membrane presentation. Induction of an anti-GCSAa secondary antibody response has been found to require the use of liposomes as GCSAa vehicle for both the priming and the boosting immunizations. In order to investigate the sensitivity of highly immune cells to the liposome presentation, immune spleen cells were stimulated in vitro with either soluble GCSAa or GCSAa-liposomes and transferred together with the immunogen into syngeneic animals. Only spleen cells from high responders, which have been immunized twice with GCSAa-liposomes, were able to generate an antibody response in naive recipients after their restimulation with the GCSAa-liposome preparation. Their restimulation with soluble antigen was ineffective unless 10% peritoneal exudate cells (PEC) from naive rats were added during the in vitro incubation. Stimulation with GCSAa-liposomes was further improved by the addition of 10% PEC. Macrophages were found to play a central role in the induction of antibody response in the recipients after stimulation with GCSAa-liposomes. Treatment of immune spleen cells with the macrophage-killing agent leucine methyl ester prior their restimulation in vitro with GCSA-liposomes in the absence of PEC, or depletion of macrophage after their in vitro incubation with this immunogen, completely abolished the induction of anti-GCSAa antibodies in the recipients.

Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs.

The immunogenicity of measles virus glycoproteins presented associated to liposomes or ISCOMs was compared with that of whole virus and solubilized membrane proteins in W/Fu rats. The rats were immunized three times at ten-day intervals with syngeneic peritoneal exudate cells fed in vitro with the various antigen preparations. A strong and persisting antibody response with haemagglutinin inhibitory and neutralization activity was observed in rats immunized with liposomes, ISCOMs, or virus. The responses were very similar despite the lower dose of protein received by rats immunized with ISCOMs (1 microgram protein) or with liposomes (20 micrograms protein). By contrast, injection of peritoneal exudate cells previously fed in vitro with soluble H and F glycoproteins resulted in only a poor and transient response. The sera from rats immunized with virus, liposomes or ISCOMs contained antibodies immunoprecipitating mainly H and F glycoproteins. Despite a strong enrichment in F polypeptides during the preparation of ISCOMs, they induced an equal anti-H and anti-F antibody response.

In vitro cellular immune response to measles viral glycoproteins: role of the antigen vector.

The capacity of measles virus and haemagglutinin (HA) and fusion (F) glycoproteins, presented either as soluble antigens, associated with liposomes or as Iscoms, to induce an in vitro primary and anamnestic cellular response was studied in syngeneic W/Fu rats using the MTT colorimetric assay. A primary cellular response was observed when the virus, HA + F liposomes or HA + F Iscoms were used as immunogens, but not when soluble HA + F glycoproteins were used. The irradiation of the naïve spleen cells at 500 rads allowed the generation of a primary response with the soluble antigens. All these primary responses were low, of similar intensity and dose-dependent. The responses were stronger after the stimulation of spleen cells from seropositive immune rats with virus, HA + F liposomes or HA + F Iscoms, whereas they were moderate after stimulation with soluble HA + F. In addition, far less antigenic material was required and the use of a vehicle for HA + F (liposomes, Iscoms) dramatically lowered the threshold of the sensitivity to the antigens. The immunization of rats with soluble HA + F glycoproteins resulted in the anergy of their spleen cells even to virus, HA + F liposomes or HA + F Iscoms. Again, irradiation of these cells could restore their ability to elicit a primary response to any type of HA + F immunogens. Using the lysosomotropic Leu-0-Met agent, all the cellular responses were found to be accessory cells dependent, the responses being restored after supplementation with 10% peritoneal exudate cells from naïve rats. These treatments did not break the immunosuppression induced by soluble HA + F glycoproteins. The uptake of the various immunogens by the murine macrophage cell line J774-1 was also studied using radioactively labelled virus and HA + F glycoproteins. The uptake of soluble HA + F was limited to 10-15%, whereas that of the other immunogens was almost complete. The data reported indicate that the modification of the supramolecular architecture of HA + F glycoproteins by their presentation in liposomes or Iscoms could modulate their immunogenicity, both qualitatively and quantitatively. It could prevent the generation of a radiosensitive suppressive mechanism, increase the sensitivity to the antigen and the activation level of the responding cell population. Quantitative modifications are accessory cell dependent and initiated within the first hour of the stimulation.

Antigen presentation by liposomes bearing class II MHC and membrane IL-1.

Liposomes containing membrane IL-1, Iak, and the antigen conalbumin were evaluated as "synthetic antigen presenting cells." The role of these three molecules in macrophage-T cell interaction was studied by testing their ability to induce the proliferation of a T-cell clone specific to conalbumin (the D10 cell line) or immune spleen cells sensitized three times in vivo with conalbumin. In the latter case, splenic macrophages were eliminated by adherence and a lysomotropic agent. The antigen conalbumin was presented on the surface of the liposomes as native undigested protein. When the liposomes presented native conalbumin, Iak, and membrane IL-1, significant proliferation occurred, but if the liposomes lacked membrane IL-1, the proliferation of the T-cell clone and the spleen cells reached only about 60 percent of the previous signal. Native conalbumin and class II antigen alone were required for T-cell activation, while membrane IL-1 only amplified the response. When the liposomes were made with only Iak and membrane IL-1, lacking conalbumin, there was no proliferation of antigen-specific target cells. These results indicated that in this synthetic system, membrane IL-1 increases the magnitude of the response but is not essential for the proliferative response of antigen-specific T cells.

Synthetic macrophages: liposomes bearing antigen, class II MHC and membrane-IL-1.

Liposomes containing membrane-IL-1, Iak, and the antigen conalbumin were evaluated as "synthetic macrophages." The role of these three molecules in macrophage-T cell interaction was studied by testing the ability of such synthetic macrophages to induce the proliferation of a T cell clone specific to conalbumin (the D10 cell line) or immune spleen cells sensitized three times in vivo with conalbumin. In the latter case, splenic macrophages were eliminated by adherence and a lysomotropic agent. The antigen conalbumin was presented on the surface of the liposomes in two forms: as native undigested protein or as a complex of Iak and naturally processed peptides isolated from peritoneal macrophages incubated with conalbumin. When the liposomes presented the Ia antigen associated with conalbumin-peptides and membrane-IL-1, the proliferation of both the D10 and the immune spleen cells was high and maximal. The proliferation response of both cell types was completely dependent on the presence of membrane-IL-1, since no proliferation occurred when liposomes were prepared with only conalbumin-peptides bound to Iak. Therefore, it could be concluded that processed antigen associated with class II MHC antigen and membrane-IL-1 were required for T cell activation. When the liposomes presented native conalbumin, Iak, and membrane-IL-1, significant proliferation occurred, but if the liposomes lacked membrane-IL-1, the proliferation of the T cell clone and the spleen cells decreased to only 40-50% of the previous signal. In this case native conalbumin and class II antigen alone were required for T cell activation, while membrane-IL-1 alone amplified the response. When the liposomes were made with only Iak and membrane-IL-1, lacking either form of conalbumin, there was no proliferation of antigen-specific target cells. These results indicated that in this synthetic system, membrane-IL-1 is essential for the proliferative response of antigen-specific T cells when conalbumin is presented as processed peptides in association with Iak.

Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release.

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.

Liposomes expressing IL 1 biological activity.

We determined the activity of IL 1, obtained from various human monocyte subcellular compartments, when associated with liposomes. Soluble IL 1, bound to the outer surface of lyophilized liposomes, stimulated responsive target cells. However, this activity was not preserved when soluble IL 1 was incorporated into the inner chambers of classical liposomes. In contrast, monocyte plasma membranes that exhibited IL 1 activity had the same level of activity when presented on lyophilized liposomes and when incorporated inside the classical liposomes. However, monocyte plasma membranes bound to the outer surface of the liposomes exhibited greater activity than the monocyte membrane IL 1 itself in its soluble form. This suggests that membrane IL 1 is an integral membrane protein, readily integrated into the lipid bilayers. Like soluble IL 1, the expression of IL 1 activity present in the cytosol of activated monocytes was decreased by incorporation into liposomes, but was high and active when presented on lyophilized liposomes. The best artificial cell reconstitution was obtained with lyophilized liposomes in association with monocyte cytosol and plasma membranes. When an unactivated monocyte compartment was mixed with one from an activated monocyte, the signal was equal to that of the activated cell compartment alone. The IL 1 activity of activated cell fractions associated with lyophilized liposomes was determined, and an increase of IL 1 activity for both plasma membranes and cytosol was observed, whereas a decrease of the signal was obtained for the lysosomal compartment. Endoplasmic reticulum showed no IL 1 activity, even after trypsin treatment. The highest activity after trypsin treatment was recovered in the cytosol associated with lyophilized liposomes, suggesting that molecules obtained after this treatment were able to bind tightly to the lipid bilayers.

Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.

Acylation of cell-associated IL-1 by palmitic acid.

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.

Dehydroepiandrosterone modulation of lipopolysaccharide-stimulated monocyte cytotoxicity.

Dehydroepiandrosterone (DHEA), the predominant androgen secreted by the adrenal cortex, can be converted to both potent androgens and estrogens. In addition to its role as a precursor for other steroid hormones, DHEA has been proposed to play an important role in immunity. This study has investigated DHEA modulation of LPS-induced monocyte cytotoxicity. Cytotoxicity markers assessed include tumor cell killing, IL-1 secretion, reactive oxygen intermediate release, nitric oxide synthetase activity as measured by the release of reactive nitrogen intermediates, complement receptor-1 cell surface protein, and TNF-alpha protein presence. Monocytes stimulated with LPS concentrations of 1.0 micrograms/ml displayed the above cytotoxic markers, whereas monocytes stimulated with DHEA alone or with LPS at a lower concentration of 0.2 ng/ml did not. However, when used simultaneously, DHEA and LPS 0.2 ng/ml displayed a synergistic effect on monocyte cytotoxicity against cancerous cell lines, IL-1 secretion, reactive nitrogen intermediate release, complement receptor-1 cell-surface protein, and TNF-alpha protein to levels comparable with levels obtained using LPS 1.0 microgram/ml. Finally, Scatchard plot analysis demonstrated the presence of a DHEA receptor in monocytes, suggesting that DHEA effects on LPS-stimulated monocytes are mediated through a receptor-dependent process.

Induction of IL-1: independent production of IL-1 alpha and IL-1 beta.

Lipopolysaccharide (LPS) either in its soluble form or associated with multilamellar phospholipid vesicles (liposomes) was investigated for its ability to induce human monocyte interleukin (IL)-1 alpha and IL-1 beta. When human monocytes were activated in vitro by LPS either in its soluble form or presented at the surface of lyophilized multilamellar vesicles (Lyo-MLV-LPS), both IL-1 alpha and IL-1 beta were detected intracellularly and extracellularly, using specific antisera. In correlation with these findings, the mRNAs for IL-1 alpha and IL-1 beta were both found by Northern blot analysis. However, when human monocytes were stimulated by LPS incorporated into multilamellar vesicles which had not been previously lyophilized, a different pattern of IL-1 protein and message was observed. IL-1 alpha activity was detected only intracellularly and not in the supernatant, while IL-1 beta was not produced at all. Northern blotting revealed only mRNA for IL-1 alpha as soon as 0.5 h after stimulation and none for IL-1 beta. These data indicate independent induction of IL-1 alpha and IL-1 beta. Moreover, it appears that the regulation occurs at the transcriptional level, since with MLV-LPS only the mRNA for IL-1 alpha was induced. The lack of IL-1 beta could be due to either a blockage at the DNA level, an undetectable level of IL-1 beta mRNA, or a very short halflife for IL-1 beta mRNA. These findings indicate that although IL-1 alpha and IL-1 beta may have identical biological properties and share the same receptor, their induction and secretion are regulated by independent pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of the melatonin receptor in human monocytes.

Earlier studies have shown that the pineal hormone melatonin activates human monocytes. It is reported here that melatonin induces the secretion of IL-1, IL-6, and TNF in fresh and 1-day in vitro cultured monocytes that also express the melatonin receptor (Kd = 270 +/- 60 pM; 42,000-48,000 receptors/cell). However, when monocytes were cultured in vitro for 2 days, the number of receptors decreased to 11,000 receptors/cell, with the same Kd. LPS activation of fresh or 1-day cultured monocytes did not result in any increase in melatonin receptor number. LPS activation of 2-day cultured monocytes led to an increase in the number of melatonin receptors, from 11,000 receptors/cell to the plateau of 42,000 to 48,000 receptors/cell. The loss of receptors by 2-day cultured monocytes was not irreversible. Melatonin did not induce the release of IL-1, TNF, or IL-6 in monocytes cultured in vitro for 3 days and for up to 15 days, and these long time cultured monocytes did not express the melatonin receptors even after activation by LPS. The loss of melatonin receptors by monocytes cultured in vitro for 3 days and for up to 15 days was irreversible. Therefore, it is shown for the first time that human monocytes express melatonin receptors. Furthermore, human monocytes express melatonin receptors differentially depending on their state of maturation, and it appears that in vitro monocyte differentiation and maturation negatively affect human monocyte melatonin receptor expression.

Cytotoxic liposomes: membrane interleukin 1 presented in multilamellar vesicles.

Paraformaldehyde-fixed lipopolysaccharide (LPS)-activated human monocytes produced significant lysis of the human melanoma cell line A375. The cytotoxic activity was retained following treatment of the fixed monocytes with anti-tumor necrosis factor (anti-TNF) antibodies but was specifically inhibited by a mixture of anti-TNF and anti-interleukin 1 (anti-IL 1) antibodies. A375 cells were also killed by plasma membranes purified from LPS-activated human blood monocytes. This activity was specifically inhibited by anti-IL 1 alpha antibodies, but only partially inhibited by anti-IL 1 beta antibodies. CHAPS detergent-extracted plasma-membrane IL 1 in its soluble form or associated with lyophilized liposomes was also able to kill A375 cells, and this antitumor activity was inhibited by anti-IL 1 antibodies. These results suggest that membrane IL 1, primarily IL 1 alpha, was cytotoxic for the A375 cells. CKS-17, a peptide synthesized with homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, when covalently bound to BSA partially inhibited the IL 1 activities of tumor cell cytotoxicity and T-cell clone proliferation, displayed by purified plasma membranes, detergent-extracted membrane IL 1, or membrane IL 1 associated with liposomes. These findings indicate that cytotoxic membrane IL 1 can be solubilized by detergent, bound to the surface of liposomes, and specifically inhibited by anti-IL 1 antibodies or the immunosuppressive peptide CKS-17.

Plasma membrane-associated tumor necrosis factor. A non-integral membrane protein possibly bound to its own receptor.

Purified plasma membranes from LPS-activated human blood monocytes produced significant lysis and growth inhibition of the TNF-sensitive L929 murine fibroblast cell line. Unactivated human monocyte plasma membranes did not display either activity. Anti-TNF serum specifically inhibited the anti-tumor activity of activated monocyte membranes whereas anti-IL-1 serum or non-specific rabbit serum decreased neither the lysis nor growth inhibition of L929 cells. Membrane-associated TNF did not behave as an integral protein as it could be eluted from the plasma membranes by either high salt or low pH treatment. Plasma membranes cleared of membrane-associated TNF by high salt treatment were able to bind TNF, and this binding was specifically inhibited by preincubation of rTNF with specific anti-TNF serum. Western blot analysis of plasma membranes showed a membrane-associated TNF with a m. w. of approximately 17 kDa present only in the activated monocytes. When the plasma membranes were preincubated with the cross-linker agent dissuccinimidyl suberate, Western blot analysis revealed the presence of a TNF-binding protein with a Mr of approximately 102 kDa. These studies indicate that unlike IL-1, membrane-associated TNF is not an integral membrane protein and that TNF may be associated with the monocyte membrane by occupying the TNF R.