Human Promonocytic Cell Line U937 Research Articles

DNA Induces Apoptosis in Electroporated Human Promonocytic Cell Line U937

Experimental gene transfer has permitted a wide variety of studies on gene regulation and function. However, possible effects of the introduced DNA on cellular metabolism are not well understood. Here we demonstrated that introduction of DNA into a promonocytic cell line, U937, by electroporation caused extensive cell death. The toxicity of DNA was concentration-dependent. Various DNAs including plasmid and genomic DNAs all caused cell death, indicating that the toxicity is nucleotide sequence-independent. DNA-induced cell death was associated with internucleosomal DNA fragmentation, a decrease in cell size, and a considerable proportion of cells outside cell cycle. From these results, we concluded that cells died by apoptosis. Our findings have experimental implication for an important issue concerning the interpretation of experiments using gene transfer. In addition, we propose that our observed phenomenon may be relevant to an important immune defense mechanism in monocytes/macrophages that facilitates a response to certain viral infections.

Phorbol ester-induced U-937 differentiation: effects on integrin alpha(5) gene transcription.

Lung injury is accompanied by increased deposition of fibronectin (FN) matrices. Activated monocytic cells recruited to sites of lung injury express integrin receptors for FN that mediate their interaction with this matrix. One such integrin, alpha(5)beta(1), mediates many of the biological effects of FN, and its expression may be important for immune cell function at sites of lung injury. Herein, we examine the expression of alpha(5)beta(1) in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) in the human promonocytic cell line U-937. We demonstrate that PMA enhanced the adherence of U-937 cells to FN by increasing the expression of both the alpha(5)- and beta(1)-subunit mRNAs and the surface expression of the protein. In U-937 cells transfected with an alpha(5) promoter-reporter gene, we found that PMA induced the transcription of the alpha(5) gene by acting on very specific promoter sequences other than activator protein-1 in a protein kinase C-dependent manner. Lipopolysaccharide had a similar effect. Modulation of alpha(5)beta(1) expression may be important for regulation of monocytic cell function in lung inflammation after injury.

Inhibition of “Tissue” Transglutaminase Increases Cell Survival by Preventing Apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of "tissue" transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca(2+)-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.

Dual Activity of Pyrrolidine Dithiocarbamate on κB-Dependent Gene Expression in U937 Cells: I. Regulation by the Phorbol Ester TPA

Pyrrolidine dithiocarbamate (PDTC) has been widely used as an inhibitor of the nuclear factor-κB (NF-κB) signalling pathway. Here, we show that κB-dependent reporter gene expression induced by low concentrations of 12- O-tetradecanoylphorbol-13-acetate (TPA) is potentiated by PDTC in the human promonocytic U937 cell line. The stimulatory effect of PDTC on κB-dependent gene expression was shown with a 4 × κB chloramphenicol acetyltransferase construct and required an intact κB element in the human immunodeficiency virus long terminal repeat (HIV-1 LTR). Unexpectedly, an HIV-1 LTR construct with a mutation of the activator protein 2 (AP-2) binding site located between the two κB elements was unresponsive to the stimulatory effect of PDTC with TPA. The stimulation or inhibition of κB-dependent gene expression was dependent on PDTC pre-treatment and the concentration of TPA. No stimulatory effect on HIV-1 LTR activity was observed with the metal chelator dipyridyl or the anti-oxidant N-acetyl- l-cysteine. These results are consistent with the hypothesis that PDTC treatment potentiated κB-dependent gene expression in a manner dependent on the concentration of TPA.

Dual Activity of Pyrrolidine Dithiocarbamate on κB-Dependent Gene Expression in U937 Cells: II. Regulation by Tumour Necrosis Factor-α

In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-κB (NF-κB) signalling pathway induced by the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-α (TNF-α)-induced NF-κB DNA binding activity but potentiated the effect of TNF-α on κB-dependent gene expression. The stimulatory effect of PDTC with TNF-α was not observed with an HIV-1 LTR reporter construct containing two mutated κB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two κB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-α, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on κB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis.

Human cytomegalovirus IE1 protein activates AP-1 through a cellular protein kinase(s).

During human cytomegalovirus (HCMV) infection, a rapid increase in AP-1 activity is detected. In this study, activation of transcription from promoters containing AP-1-binding sites by the IE1 protein of HCMV was examined. In transient transfection assays with reporter plasmids, it was found that IE1 strongly induced AP-1-driven transcription. Cells stably expressing IE1 also showed higher levels of AP-1 activity than did control cells. IE1 expression did not raise levels of c-jun and c-fos RNA, as determined by quantitative RT-PCR. AP-1 induction by IE1 was blocked efficiently by protein kinase inhibitors in a cell type-dependent manner; for example, by staurosporine in the human microglial cell line U373MG and by H7 in the human promonocytic cell line U937. IE1-driven activation of AP-1 was increased dramatically by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1). The results of this study indicate that IE1 activates AP-1 at the post-transcriptional level and that MEKK1 may play an important role in this process.

Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the tumor necrosis factor alpha signalling pathway.

The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-alpha). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-alpha treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642-1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-alpha treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors.

Sensitivity of Leishmania viannia panamensis to pentavalent antimony is correlated with the formation of cleavable DNA-protein complexes.

The emergence of Leishmania less sensitive to pentavalent antimonial agents (SbVs), the report of inhibition of purified topoisomerase I of Leishmania donovani by sodium stibogluconate (Pentostam), and the uncertain mechanism of action of antimonial drugs prompted an evaluation of SbVs in the stabilization of cleavable complexes in promastigotes of Leishmania (Viannia). The effect of camptothecin, an inhibitor of topoisomerase, and additive-free meglumine antimoniate (Glucantime) on the stabilization of cleavable DNA-protein complexes associated with the inhibition of topoisomerase was assessed in the human promonocytic cell line U-937, promastigotes of L. (Viannia) panamensis selected for SbV resistance in vitro, and the corresponding wild-type strain. The stabilization of cleavable complexes and the 50% effective dose (ED50) of SbVs for parasites isolated from patients with relapses were also evaluated. The median ED50 for the wild-type strain was 16. 7 microg of SbV/ml, while that of the line selected for resistance was 209.5 microg of SbV/ml. Treatment with both meglumine antimoniate and sodium stibogluconate (20 to 200 microg of SbV/ml) stabilized DNA-protein complexes in the wild-type strain but not the resistant line. The ED50s of the SbVs for Leishmania strains from patients with relapses was comparable to those for the line selected for in vitro resistance, and DNA-protein complexes were not stabilized by exposure to meglumine antimoniate. Cleavable complexes were observed in all Leishmania strains treated with camptothecin. Camptothecin stabilized cleavable complexes in U-937 cells; SbVs did not. The selective effect of the SbVs on the stabilization of DNA-protein complexes in Leishmania and the loss of this effect in naturally resistant or experimentally derived SbV-resistant Leishmania suggest that topoisomerase may be a target of antimonial drugs.

Rooperol tetraacetate decreases cytokine mRNA levels and binding capacity of transcription factors in U937 cells.

We have previously described inhibition of the synthesis of three acute-phase inflammatory cytokines in human and rat macrophages by acetate esters of rooperol, a dicatechol of plant origin. Analysing the mechanism of anticytokine activity of rooperol, we compared levels of TNFalpha, IL-1beta and IL-6 mRNAs in the human promonocytic U937 cell line pretreated with phorbol myristate acetate (PMA) and incubated with rooperol tetraacetate (RTA) alone or in combination with LPS (500 ng/ml). It was found that 10 microM RTA decreased the levels of cytokine mRNAs both in the presence and absence of LPS, suggesting pretranslational inhibition of cytokine synthesis. Electrophoretic mobility shift analysis (EMSA) showed that RTA may influence cytokine mRNA expression by decreasing the binding activity of transcription factors NF-kappaB and AP-1.

Histamine modulates the expression of c-fos through cyclic AMP production via the H2 receptor in the human promonocytic cell line U937.

We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors.

Constitutive Activation of U937 Promonocytic Cell Clones Selected for Their Resistance to Parvovirus H-1 Infection

The human promonocytic cell line U937 is highly sensitive to the lytic effect of the autonomous parvovirus H-1. Rare cell variants that resisted H-1 virus infection could be isolated, of which four (RU1, RU2, RU3, and RU4) were further characterized. In contrast to parental cells, the RU clones sustained an abortive H-1 virus infection. Three of the clones showed a significant decrease in the accumulation levels of the c-Myc oncoprotein and in their capacity for forming tumors in immunodeficient mice. Surprisingly, all RU clones resisted the suppressing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on c-myc oncogene expression and cell proliferation. In contrast, RU clones exhibited the TPA-induced changes in membrane surface antigens and nonspecific esterase activities that are characteristic of monocytic differentiation. Studies of the activation steady-state of RU cells demonstrated the constitutive production of significant amounts of nitric oxide (NO) and superoxide anion (O-.2). Inhibitors of NO and O-.2, production sensitized all RU cells to the killing effect of parvovirus H-1 and increased the production of infectious viral particles. These data argue for the participation of active oxygen species in macrophage defence mechanisms against parvovirus infection. Moreover, the use of parvovirus H-1 as a selective agent in a cell-colony formation assay allowed us to show that expression of defined markers of monocytic differentiation can be uncoupled from suppression of proliferation.

Constitutive Activation of U937 Promonocytic Cell Clones Selected for Their Resistance to Parvovirus H-1 Infection

AbstractThe human promonocytic cell line U937 is highly sensitive to the lytic effect of the autonomous parvovirus H-1. Rare cell variants that resisted H-1 virus infection could be isolated, of which four (RU1, RU2, RU3, and RU4) were further characterized. In contrast to parental cells, the RU clones sustained an abortive H-1 virus infection. Three of the clones showed a significant decrease in the accumulation levels of the c-Myc oncoprotein and in their capacity for forming tumors in immunodeficient mice. Surprisingly, all RU clones resisted the suppressing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on c-myc oncogene expression and cell proliferation. In contrast, RU clones exhibited the TPA-induced changes in membrane surface antigens and nonspecific esterase activities that are characteristic of monocytic differentiation. Studies of the activation steady-state of RU cells demonstrated the constitutive production of significant amounts of nitric oxide (NO) and superoxide anion (O−2⋅ ). Inhibitors of NO and O−2⋅ . production sensitized all RU cells to the killing effect of parvovirus H-1 and increased the production of infectious viral particles. These data argue for the participation of active oxygen species in macrophage defence mechanisms against parvovirus infection. Moreover, the use of parvovirus H-1 as a selective agent in a cell-colony formation assay allowed us to show that expression of defined markers of monocytic differentiation can be uncoupled from suppression of proliferation.

Nuclear Factor kB Is Activated by Arachidonic Acid but Not by Eicosapentaenoic Acid

The Ω-6 arachidonic acid supplementation of the human promonocytic cell line U937 strongly stimulates the nuclear translocation of the transcription factor NF-kB. Inhibitors of arachidonate oxidative metabolism prevent NF-kB activation, indirectly indicating a role for prostaglandin and leukotriene metabolites in the genesis of this phenomenon. Of note, Ω-3 eicosapentaenoic acid does not exert any effect on NF-kB DNA binding. In subsequent experiments, prostaglandin E2consistently showed the ability to activate NF-kB in U937 promonocytic cells, as well as in J774 macrophages. NF-kB activation by arachidonate, together with the lack of effect by eicosapentaenoic acid, suggests a way to modulate the expression of certain genes by means of a suitable dietary n-6/n-3 fatty acid ratio.

HIV-regulated diphtheria toxin A chain gene confers long-term protection against HIV type 1 infection in the human promonocytic cell line U937.

Gene therapy approaches have recently been investigated for the treatment of acquired immunodeficiency syndrome (AIDS), both in preclinical and clinical studies, because more traditional antiviral agents have proven to be of limited effectiveness. We have previously shown that long-term protection against both laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) was conferred by HIV-regulated diphtheria toxin A (DT-A) chain in a human T cell line. Because the monocyte/macrophage cell is an important reservoir for HIV-1 in infected individuals, we sought here to determine whether HIV-regulated DT-A would also be effective in the promonocytic cell line U937. We report here that long-term protection, conferred by HIV-regulated DT-A, was observed in U937 cells, but that protection was dependent on the stock of HIV IIIB used for challenge. HIV production was measured by p24 assays, polymerase chain reaction (PCR) for HIV vif, gag, and reverse transcriptase (RT) sequences, and cocultivation with peripheral blood mononuclear cells (PBMCs). Complete protection was seen in DT-A-transduced cells with a stock of IIIB propagated on H9 cells and titered on peripheral blood mononuclear cells (PBMCs), while protection in these same cells with a second stock of IIIB, propagated and titered on H9 cells, was only partial and dose dependent.

Poly(ADP-Ribose) Polymerase-Activity in Various U937 Cell Subclones with Different Susceptibility to HIV-1 Infection - Its Dramatic Decrease Following Persistent Virus-Infection

Poly(ADP-ribose) polymerase, a nuclear enzyme, is suggested to be involved in apoptotic cell death. It is also known that apoptotic cell death following HIV-1 infection is the most important feature of AIDS pathogenesis. Thus, to evaluate the relations between the enzyme and HIV-1 infection, we examined the enzyme activity of several subclones of human promonocytic cell line U937, which showed different susceptibility to HIV-1 infection. The nuclear extracts of two "high type clones" (possessing high susceptibility to HIV-1 infection) contained approximately 4 to 7-fold less enzyme than two low type clones when assayed under a full activation of enzyme. Parent clone, possessing an intermediate susceptibility to HIV-1, showed an intermediate enzyme level, suggesting that low level of this enzyme in cells is important for an effective infection of HIV-1. Furthermore, when these U937 subclones persistently infected with HIV-1 were examined, a dramatic decrease of the enzyme activity, reaching 2 to 16% of uninfected cells, was observed in all of these clones. The levels of poly(ADP-ribose) glycohydrolase in these clones were relativity unchanged. Activity gel analysis and immunoblotting of the enzyme in the clones revealed that the low enzyme activities observed in uninfected "high type clones" and all HIV-1-infected clones were due to a marked decrease of the enzyme protein itself. All of these results suggest that HIV-1 infection involves some mechanism to downregulate cellular poly(ADP-ribose) polymerase and that a lower level of the enzyme may be essential for an effective production of the virus and/or for a stable virus/host interaction.

Viral activation from latency during retrodifferentiation of U937 cells exposed to phorbol ester followed by infection with human immunodeficiency virus type 1

To determine the mechanism underlying the human immunodeficiency virus type 1 (HIV-1) latency and its activation in monocyte/macrophage lineage, the human promonocytic cell line U937 was infected with HIV-1 after differentiation with varied doses of phorbol 12-myristate 13-acetate (PMA). Variously differentiated intermediate stages were generated in U937 cells in a dose-dependent manner. When these cells were infected with lymphotropic HIV-1, the kinetics of the production of HIV-1 DNA, the appearance of HIV-1 antigen-positive cells, and viral production in the conditioned media were slower at higher doses of PMA. This different susceptibility to the infection was not due to the rate of HIV-1 adsorption. Viral replication from latency in the differentiated cells was activated in proportion with the retrodifferentiation observed in long-term cultures of the host cells. Thus, our data demonstrate the close correlation between the regulation of HIV-1 replication and the differentiation stage of monocyte/macrophage lineage cells at the time of HIV-1 infection. The retrodifferentiation phenomenon in infected cells seems to be particularly important for understanding the mechanisms for HIV-1 activation from latency.

Binding of lactoferrin and transferrin to the human promonocytic cell line U937. Effect on iron uptake and release.

We have compared the ability of lactoferrin and transferrin to interact with and donate iron to the monocytic cell line U937. About 10 times more lactoferrin was bound than transferrin, but most lactoferrin bound nonspecifically, and the degree of specific binding was similar for both proteins (2-3 x 10(6) sites/cell). The binding affinity for lactoferrin (83 nM) was about 4-fold lower than for transferrin (21 nM). Lactoferrin did not inhibit binding of transferrin, or vice versa. Binding of lactoferrin was not inhibited by 30 mM glucose or fucose nor by incubating the cells with heparinase. Transferrin, but not lactoferrin, was internalized, and 3 mM primaquine caused intracellular accumulation of transferrin but not lactoferrin. The cells rapidly acquired iron from transferrin, but uptake from lactoferrin was 10-fold slower and probably resulted from transfer of 59Fe from lactoferrin to unlabeled transferrin during culture. Lactoferrin, but not transferrin, released iron to the extracellular medium when bound to U937 cells. Lactoferrin inhibited cellular uptake of iron from Fe-nitrilotriacetate but not from transferrin. It is concluded that transferrin, but not lactoferrin, acts as an iron donor to U937 cells. Lactoferrin may regulate uptake of potentially toxic non-transferrin-bound iron.

Effect of heat treatment and other milk proteins on the interaction of lactoferrin with monocytes

The interaction of lactoferrin from human and bovine milk with the human promonocytic cell line U937 has been studied. Both human and bovine Fe-lactoferrins bound to the cells. Binding of bovine lactoferrin was inhibited by excess bovine lactoferrin but not by human lactoferrin, suggesting that the binding mechanisms for the two proteins are different. Binding of human but not bovine lactoferrin was inhibited by bovine lactoperoxidase, while a 20-fold excess of human IgA inhibited binding of human but not bovine lactoferrin. Human and bovine alpha-lactalbumins, bovine beta-lactoglobulin, and human lysozyme had no effect on binding of lactoferrin from either species. Samples of bovine Fe- and apolactoferrin in capillary tubes were exposed to temperatures of 72 degrees C for 20 s, 85 degrees C for 20 min or 137 degrees C for 8 s. All the heated samples inhibited binding of native Fe- and apolactoferrin, though to a lesser extent than the native proteins. Both heated and native lactoferrins enhanced [3H]thymidine incorporation by U937 cells, except for Fe-lactoferrin heated at 85 degrees C for 20 min, which was inhibitory. These results suggest that heat treatment of lactoferrin under conditions used for industrial processing does not greatly affect its ability to interact with and stimulate monocytic cells, and that other milk proteins in general do not interfere with lactoferrin-monocyte interactions. It may thus be feasible to incorporate biologically active lactoferrin into infant formulas.

Tumor necrosis factor cytotoxicity is associated with phospholipase D activation

The activation of phospholipase D in different cell lines treated with recombinant human tumor necrosis factor (TNF) has been investigated. When the murine fibrosarcoma cell lines L929 and WEHI164c113, as well as the human promonocytic cell line U937, were prelabeled with [14C]palmitic acid or [3H]arachidonic acid, and treated with TNF in the presence of ethanol, TNF induced the synthesis of [14C]phosphatidylethanol or [3H]phosphatidylethanol, respectively, as the result of a phospholipase-D-catalyzed transphosphatidylation. TNF-induced phospholipase D activity was observed 1 h before the onset of cell killing and gradually increased thereafter. Subclones selected for resistance to TNF cytotoxicity did not show phospholipase D activation in response to TNF. In contrast, when these subclones were treated with TNF in the presence of actinomycin D, TNF cytotoxicity as well as TNF-induced phospholipase D activity could be restored. TNF cytotoxicity and TNF-induced phospholipase D activity were equally modulated by various drugs known to interfere with different steps in the TNF-signaling pathway. Phospholipase D activation was found not to be the result of cell killing per se, as a number of other cytotoxic reagents were unable to activate phospholipase D. Prelabeling of cells with [14C]lysophosphatidylcholine indicated phosphatidylcholine as one of the substrates for TNF-activated phospholipase D. In conclusion, this study demonstrates that TNF-induced cytotoxicity is associated with activation of phospholipase D.

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.