LMP-420: a novel purine nucleoside analog with potent cytotoxic effects for CLL cells and minimal toxicity for normal hematopoietic cells.

B-cell chronic lymphocytic leukemia (CLL) is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 h was 245 n. LMP-420-induced time- and dose-dependent apoptosis, as shown by annexin V staining, caspase activation and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL and Bcl-2. CLL cells from patients with poor prognostic indicators showed LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of nuclear factor-kappaB and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.

Covalent complexes of antigen and alpha(2)-macroglobulin: evidence for dramatically-increased immunogenicity.

A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.

Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections.

Studies of the effects of retroviruses on the immune system, which date back through thirty years of investigations, are reviewed. In the earliest published studies in the 1960s, it was demonstrated that mice infected with oncogenic viruses were immunosuppressed. Since then, numerous articles have been published describing profound immunodeficiencies observed in vivo in humans infected with human immunodeficiency virus and in animals such as cats infected with the feline immunodeficiency virus. In vitro investigations have shown that inactivated retroviruses or transmembrane envelope protein p15E as well as a synthetic 17-amino acid peptide (CKS-17) impressively conserved within the transmembrane envelope protein of several animal or human retroviruses are highly immunosuppressive. More recently, dysfunction of cytokines produced by CKS-17 at both a cellular and molecular level have been found to mimic influences observed in vivo in patients infected with the human immunodeficiency virus. CKS-17 has also been shown to induce cAMP in vitro. The significance of these observations to understanding the immunological disturbances observed in malignancy, cytokine biosynthesis, and modulations of immune functions through cAMP is discussed.

Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway.

HIV-1 Nef protein shares a significant homology with the immunosuppressive and highly conserved retroviral transmembrane protein p15E. In the present study, extracellular Nef protein is shown to induce interleukin (IL)-10 mRNA expression in human peripheral blood mononuclear cells as well as in cells of H9 T and U937 promonocytic human cell lines. Release of IL-10 protein into supernatants of peripheral blood mononuclear cells stimulated with Nef is dose-dependent. Expression of cytokines IL-2, IL-4, IL-5, IL-12 p40, IL-13, and interferon gamma is not affected by Nef stimulation. IL-10 protein production induced by Nef is inhibited by the calcium/calmodulin phosphodiesterase inhibitor W-7 but not by the protein kinase A inhibitor H-89 nor the protein kinase C inhibitors staurosporine and calphostin C. The calcium chelating agent EGTA also inhibits the IL-10 production induced by Nef, and this inhibition is reversed by the addition of calcium along with Nef. These findings indicate that extracellular Nef may contribute to the immunopathogenesis of HIV infection by inducing IL-10.

Induction of intracellular cAMP by a synthetic retroviral envelope peptide: a possible mechanism of immunopathogenesis in retroviral infections.

A synthetic heptadecapeptide, CKS-17, represents the highly conserved amino acid sequences occurring within the transmembrane envelope protein of many animal and human retroviruses. CKS-17 has been demonstrated to exhibit suppressive properties for numerous immune functions. We have recently shown that CKS-17 acts as an immunomodulatory epitope causing an imbalance of human type 1 and type 2 cytokine production and suppression of cell-mediated immunities. cAMP, an intracellular second messenger, plays an important role in regulation of cytokine biosynthesis--i.e., elevation of intracellular cAMP levels selectively inhibits type 1 cytokine production but has no effect or enhances type 2 cytokine production. Here, we demonstrate that CKS-17 induces dramatic rises in the intracellular cAMP levels of a human monocyte cell line and of human peripheral blood mononuclear cells in a time- and dose-dependent manner. A peptide corresponding to the reverse sequence of CKS-17, used as control, has no effect on intracellular cAMP levels. The cAMP-inducing ability of CKS-17 is significantly blocked by SQ-22536, an inhibitor of adenylate cyclase. These results indicate that CKS-17, a highly conserved component of the transmembrane proteins of immunosuppressive retroviruses, induces increased intracellular levels of cAMP via activation of adenylate cyclase and suggest that this retroviral envelope peptide may differentially modulate type 1 and type 2 cytokine production through elevation of intracellular cAMP levels.

Monocyte-induced cytokine expression in cultured human retinal pigment epithelial cells.

Monocytes and retinal pigment epithelial cells are intimately associated in membranes of eyes with proliferative vitreoretinopathy and in certain types of uveitis. The goal of this study was to determine whether monocytes modulate cytokine expression in retinal pigment epithelial cells, and if so, to identify the monocyte products responsible for this effect. Cultured human retinal pigment epithelial cells were exposed to varying concentrations of monocyte-conditioned medium from unstimulated human monocytes for 1-48 hr, or from monocytes prestimulated with lipopolysaccharide. mRNA expression of interleukin-1 beta, interleukin-6, interleukin-8, melanoma growth stimulating activity/gro alpha and gamma, macrophage colony stimulating factor, transforming growth factor-beta 2, basic fibroblast growth factor and activin beta A chain was determined by reverse transcription polymerase chain reaction. Protein secretion of selected cytokines, interleukin-1 beta, interleukin-6, interleukin-8, macrophage colony stimulating factor and transforming growth factor-beta 2 was measured in RPE-conditioned medium by ELISA. Retinal pigment epithelial cells constitutively expressed mRNA for interleukin-6, macrophage colony stimulating factor, transforming growth factor-beta 2, basic fibroblast growth factor and activin beta A chain. Interleukin-1 beta, melanoma growth stimulating activity/gro alpha and gamma and interleukin-8 were not expressed under basal conditions. Stimulated monocyte-conditioned medium markedly induced mRNA of all cytokines except basic fibroblast growth factor and transforming growth factor-beta 2 in a dose- and time-dependent manner. Unstimulated monocyte-conditioned medium was a less potent inducing agent, but still enhanced mRNA expression of interleukin-6, interleukin-8 and melanoma growth stimulating activity/gro alpha. Stimulated monocyte-conditioned medium also induced a time-dependent increase in interleukin-6, Interleukin-8, macrophage colony stimulation factor and transforming growth factor-beta 2, but not interleukin-1 beta protein secretion (p < 0.05 for all time points). Neutralizing antibodies to interleukin-1 beta, or tumour necrosis factor alpha, but not interleukin-1 alpha, significantly reduced cytokine mRNA expression induced by stimulated monocyte-conditioned medium. The combination of all three neutralizing antibodies almost entirely eliminated monocyte-induced mRNA expression and protein production of all cytokines studied. Activated monocytes secrete a heterogeneous mixture of products that together strongly induce expression of multiple cytokines in human retinal pigment epithelial cells. Most if not all of the inducing effect can be accounted for by interleukin-1 beta and tumour necrosis factor alpha. Because cytokines have been implicated in proliferative vitreoretinopathy and uveitis, monocyte-mediated cytokine expression by RPE cells may serve to initiate and perpetuate these diseases.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

Transcriptional down-regulation of tumor necrosis factor-alpha gene expression by a synthetic peptide homologous to retroviral envelope protein.

We have previously shown that a synthetic peptide (CKS-17) homologous to retroviral envelope protein suppresses the accumulation of superantigen staphylococcal enterotoxin-induced TNF-alpha mRNA in human PBMC and in highly purified human monocytes. The present study was designed to examine the underlying mechanism(s) by which CKS-17 down-regulates the TNF-alpha mRNA expression using a human acute monocytic leukemia cell line THP-1 stimulated with the superantigen staphylococcal enterotoxin E. A cyclooxygenase inhibitor indomethacin does not reverse the inhibition of TNF-alpha mRNA expression by CKS-17, suggesting that prostaglandins are not responsible for the suppressive action of CKS-17. The inhibitory effect of CKS-17 is, however, significantly blocked by a protein synthesis inhibitor cycloheximide, indicating that CKS-17 requires de novo protein synthesis to induce the suppressive activity. The mRNA stability assays using actinomycin D show that CKS-17 does not decrease the TNF-alpha mRNA stability. Nuclear run-on transcription assays further reveal that CKS-17 suppresses the TNF-alpha mRNA transcription rate. Taken together, these results suggest that the synthetic retroviral peptide CKS-17 down-regulates TNF-alpha mRNA expression through inhibition of transcriptional activation of the TNF-alpha gene, which requires de novo synthesis of a transcriptional repressor protein(s).

A synthetic peptide homologous to retroviral envelope protein down-regulates TNF-alpha and IFN-gamma mRNA expression.

We investigated the influence of CKS-17, a synthetic heptadecapeptide that corresponds to a highly conserved domain of the immunosuppressive retroviral envelope protein p15E, on staphylococcal enterotoxin B (SEB)-induced TNF-alpha gene expression in human peripheral blood mononuclear cells and highly purified human monocyte preparations, as well as the production of TNF-alpha protein, using human peripheral blood mononuclear cells. RNA hybridization studies show that CKS-17 inhibits SEB-induced TNF-alpha mRNA accumulation in human peripheral blood mononuclear cells and human monocytes. CKS-17 is also shown to be highly suppressive for SEB-induced production of TNF-alpha proteins. Similarly, CKS-17 inhibits expression of SEB-induced IFN-gamma mRNA in human peripheral blood mononuclear cells. These results suggest that CKS-17 down-regulates both TNF-alpha and IFN-gamma production at mRNA level.

Suppression of human interferon-gamma production by a 17 amino acid peptide homologous to the transmembrane envelope protein of retroviruses: evidence for a primary role played by monocytes.

CKS-17, a synthetic amino acid peptide homologous to a highly conserved region of retroviral transmembrane protein exerts a suppressive action on staphylococcal enterotoxin A (SEA)-induced the production of IFN-gamma by human peripheral blood mononuclear cells (PBMC) (Ogasawara et al., J. Immunol. 141, 615, 1988). This action has been shown in the present study to be preceded by dramatic clustering of PBMC. Clusters appear within 3 hr of exposure of PBMC to CKS-17; they are dose dependent, inhibited by cycloheximide, and require a temperature of 37 degrees C. The cells in the clusters are predominantly monocytes. Although it has been previously shown that CKS-17 inhibits monocyte-mediated killing by inactivating IL-1 (Kleinerman et al., J. Immunol. 139, 2329, 1987) and production of IL-2 by murine thymoma cells treated with IL-1 (Gottlieb et al., J. Immunol. 142, 4321, 1989), in the present study we show that IL-1 does not prevent clustering of PBMC by CKS-17. Using CKS-17 and highly purified monocytes or lymphocytes, profound alterations occur only with monocytes, as revealed by light or electron microscopy. SEA- or staphylococcal enterotoxin B-induced production of IFN-gamma is inhibited when highly purified monocytes pretreated with CKS-17 are cocultured with highly purified T lymphocytes. Thus, CKS-17 induces dramatic clustering of cells apparently by inducing alterations of monocytes but not lymphocytes, suggesting that CKS-17 may interfere with the capacity of monocytes to facilitate production of IFN-gamma by T lymphocytes.

Specific association of retroviral envelope protein, p15E, with human cell surfaces.

Experiments were carried out to analyze the binding sites on human cells for highly purified retroviral protein p15E isolated from Feline Leukemia Virus, Rickard Strain. Binding of 125I-labeled p15E was tested with surfaces of human peripheral blood lymphocytes and 3 cell lines, Raji, MOLT-4, and U-937. 125I-labeled p15E showed specific binding to human peripheral blood lymphocytes. In addition, all of the cell lines tested showed binding of 125I-labeled p15E. Using U-937 cells, we characterized the interaction between p15E and the surface of these cells, and showed that the binding was specific by the following 3 different sets of evidence: (i) in equilibrium binding experiments, 18,000 binding sites with a dissociation constant of 2 x 10(-9) M were present on U-937 cells; (ii) trypsin or N-glycanase treatment decreased the binding sites of 125I-labeled p15E; and (iii) by affinity chromatography using p15E or BSA Sepharose columns, the isolated membranes of 125I-labeled U-937 cells previously treated with Triton X-100 showed a significantly higher binding to the p15E column than to the BSA column.

The suppressive effect of a synthetic retroviral peptide on the human IFN gamma production is abrogated by the combined stimulation with IL-1 and IL-2.

Certain retroviral envelope proteins and peptides have been shown to be highly immunosuppressive. Recently, we have demonstrated that a synthetic 17 amino acid peptide (CKS-17*) homologous to a highly conserved region in the transmembrane portion of the envelope of several human or animal retroviruses suppresses the production of human interferon-gamma (IFN gamma) by human peripheral blood leukocytes (PBL). In the present investigation, we studied the role of exogenous IL-1 or IL-2, and IL-1 plus IL-2 on the suppressive action of CKS-17* in the production of IFN gamma. The results showed that preculture of PBL with CKS-17* reduced the production of IFN gamma in a dose-dependent manner. The addition of IL-1 or IL-2 reduced, in part, this suppression of IFN gamma production. Full abrogation of the inhibition attributable to CKS-17, however, occurred only when PBL precultured with CKS-17* were recultured with staphylococcus enterotoxin A (SEA) together with exogenous IL-1 plus IL-2. These results show that the inhibition of IFN-gamma production by CKS-17* is reversible. The findings indicate that cytokines can modulate certain of the immunosuppressive actions attributable to retroviruses or their components and suggest that some cytokines influence immunosuppressive consequences of retroviral infection.

A synthetic peptide homologous to retroviral transmembrane envelope proteins depresses protein kinase C mediated lymphocyte proliferation and directly inactivated protein kinase C: a potential mechanism for immunosuppression.

CKS-17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS-17-BSA). To define its site of action, we determined if CKS-17 conjugated to human serum albumin (CKS-17-HSA) could block the direct activation of lymphocytes by phorbol-12-myristate-13-acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiC8). CKS-17-HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose-dependent manner with up to 88% inhibition occurring with 15 microM CKS-17-HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 microM CKS-17-HSA. Based on these findings we investigated the effect of CKS-17-HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS-17-HSA caused a dose-dependent enzyme inhibition with a concentration giving half-maximal inhibition (IC50) of ca.3 microM and greater than 95% inhibition at 15 microM CKS-17-HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS-17-HSA did not inhibit the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PK-A) nor the calcium and phospholipid-independent form of PKC (PK-M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA-stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS-17-HSA may be due to the direct inactivation of PKC.

Inhibition of protein kinase C by a peptide conjugate homologous to a domain of the retroviral protein p15E.

Retroviral infection is associated with immunosuppression, which has been shown to be due, in part, to the action of the envelope protein p15E. We studied a synthetic peptide (CKS-17) homologous to a highly conserved domain of the retroviral envelope protein p15E, which, when conjugated to BSA (CKS-17-BSA), can inhibit IL-1- and phorbol ester-mediated responses in cultured murine thymoma cells, and Ca2(+)- and phosphatidylserine-dependent protein kinase C (PKC) activity of cell homogenates. We characterized the mechanism of inhibition of PKC by the peptide. Using PKC purified from rat brain we found that CKS-17-BSA inhibited PKC-catalyzed Ca2(+)- and phosphatidylserine-dependent histone phosphorylation with an estimated ID50 of 4 microM. CKS-17-BSA did not inhibit the catalytic subunit of cAMP-dependent protein kinase. CKS-17-BSA also inhibited the Ca2(+)- and PS-independent activity of a catalytic fragment of PKC that was generated by limited trypsin treatment. However, CKS-17-BSA did not act as a competitive inhibitor of PKC with respect to ATP or phosphoacceptor substrate, despite the similarity between the CKS-17 sequence and substrates and pseudosubstrates of PKC. We conclude that this peptide homologue of a retroviral envelope protein has a novel mechanism of inhibition of PKC.

A soluble inhibitor of T lymphocyte function induced by HIV-1 infection of CD4+ T cells: characterization of a cellular protein and its relationship to p15E.

Soluble suppressor factor (SSF), first described in association with HIV-1 infection in vivo, is a molecule(s) capable of inhibiting T cell-dependent immune reactivity. Its relationship to human immunodeficiency virus (HIV) was further defined as supernatants of mononuclear cell cultures from HIV-1-seropositive carriers, CD4+ T lymphocytes infected with HIV-1 in vitro, and a T cell hybridoma incorporating CD4+ lymphocytes from an HIV-1-seropositive individual were shown to elaborate factors with similar activity profiles. These factors were recognized antigenically by certain antibodies directed against epitopes of p15E, a transmembrane protein of murine leukemia virus which shares regions of identity with proteins deduced from human endogenous retroviral envelope transcripts as well as HIV. These reagents precipitated a single-chain, nonglycosylated, nonviral protein of molecular weight 57,000 Da from SSF-producing cells. There was no cross-reactivity with antisera recognizing the IL-2R alpha-chain (CD25) or tumor necrosis factor. This molecule was present in very low levels in PHA-activated T lymphocytes and was upregulated following their infection with HIV-1. Isolation of HIV-linked SSF should permit comparisons with other virion, cellular, and serum inhibitory substances described in AIDS, and perhaps suggest therapeutic strategies.

Inhibition of murine cytotoxic T lymphocyte activity by a synthetic retroviral peptide and abrogation of this activity by IL.

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the generation of murine alloantigen-specific CTL activity in vitro. CKS-17 coupled to a carrier protein, BSA or human serum albumin, inhibited the generation of anti-allo CTL in a dose-dependent manner. Controls consisting of BSA and human serum albumin, which had undergone the coupling procedure or neurotensin, an unrelated peptide, coupled to BSA in an identical manner as CKS-17 showed no such inhibitory action. The suppression was not restricted to the Ag specificity of the CTL activity. CKS-17 exerted inhibitory effects on the early afferent phase of CTL induction. Kinetic studies showed that the suppressive activity occurred when CKS-17 was introduced to the immunologically stimulating culture concomitant with or up to 48 h after initiation of culture. Analysis of the frequency of CTL precursor cells using limiting-dilution assays revealed that CKS-17 did act to reduce the number of precursor cells. Abrogation of the inhibition of CTL activity was observed when IL-2 was introduced to the culture together with the stimulator cells. Other lymphokines, such as IL-4, exerted a similar influence to counteract this suppression.

Synthetic peptide corresponding to a conserved domain of the retroviral protein p15E blocks IL-1-mediated signal transduction.

We studied the mode of action of the synthetic peptide CKS-17, which is a heptadecapeptide homologous to a highly conserved region of the immunosuppressive retroviral envelope protein p15E, as well as to envelope proteins of the human T cell leukemia virus I and II. Previous studies have established that CKS-17 conjugated to BSA (CKS-17-BSA) inhibited IL-1-mediated tumor toxicity in melanoma cells and proliferation in murine Th clones. We examined the effects of CKS-17-BSA on IL-1 action. CKS-17-BSA did not bind to IL-1, nor did it affect the number of IL-1 receptors, their binding affinity, or their ability to internalize IL-1. However, CKS-17-BSA inhibited production of IL-2 by murine thymoma cells treated with IL-1 or with 12-O-tetradecanoyl phorbol-13 acetate. The potent protein kinase C inhibitor, H7, also inhibited IL-1-mediated responses, while HA1004, a weak inhibitor of protein kinase C, did not. Protein kinase C activity in the cytosolic fraction prepared from thymoma cells was found to be inhibited by CKS-17-BSA in a dose-dependent manner. All of these findings are consistent with the idea that CKS-17-BSA inhibits IL-1-mediated responses by interfering with signal transduction through a protein kinase C pathway.

Production of a retroviral P15E-related chemotaxis inhibitor by IL-1-treated endothelial cells. A possible negative feedback in the regulation of the vascular response to monokines.

The effects of IL-1 on vascular endothelium result in a complex set of alterations which are potentially disruptive of vessel wall and underlying tissue integrity. The present study was aimed at investigating possible regulation of such potentially destructive responses elicited by IL-1 on endothelial cells. Culture supernatants of IL-1-treated human umbilical vein endothelial cells (HEC) were depleted of retroviral p15E-related Ag with immobilized anti-p15E mAb. The monocyte chemotactic and polarizing activity of supernatants of IL-1-treated HEC (presumably related to colony-stimulating factors being released by HEC) was markedly augmented by absorption on immobilized anti-p15E antibodies. Irrelevant IgG had no effect and anti-p15E antibodies did not affect the chemotactic activity of supernatants from unstimulated HEC. The material eluted from Sepharose-bound anti-p15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. The alpha and beta molecular species of IL-1 were equally effective in inducing the production of p15E-related inhibitor. The production of a p15E-related inhibitor of chemotaxis induced by IL-1 in HEC may represent a negative signal in the regulation of the potentially destructive responses to pro-inflammatory cytokines.