Glass-adherent Cells Research Articles

Mechanosensetive ATP Release Involves a Non-Conductive Pathway: Evidence from Large Field of View Real-Time Imaging

Cell swelling is a potent ATP secretion stimulus. While the sources of ATP are known, it's mechanisms of secretion in epithelial cells remain unclear. Two secretion pathways have been proposed: exocytosis of nucleotide-containing vesicles or conductive release through pore-forming channels, like pannexins or connexins. In this project, we have tested whether ATP is released or not through a conductive pathway in A549 lung epithelial cells. Real-time ATP secretion from glass-adherent A549 cells was imaged with a custom-designed lens system, combining a large field of view and a high light-gathering power, mounted directly on a Photometrics® EvolveTM 512 EMCCD camera. ATP was detected with a light-emitting luciferin-luciferase reaction. Secretion was stimulated by acute cell swelling or exposure to ionomycin (10 µM). Plasma membrane permeability to large molecules was assessed in parallel by propidium iodide (PI) uptake with epifluorescence Zeiss Axio Observer microscope. Cells were grown in 10% (control cells) or 0.1% FBS (fragile cells). Our novel imaging system revealed a strong homogeneous ATP secretion lasting several minutes from control A549 cells stimulated with 50% hypotonic shock. Yet, no PI-stained cells were detected under these conditions. In contrast, cells rendered fragile, and thus prone to cell lysis, showed an appreciable amount of PI-stained cells post-hypotonic shock and a punctuate pattern of ATP secretion co-localizing with PI-positive cells. Sudden intracellular calcium elevation with ionomycin provoked a rapid, albeit small, ATP secretion. Based on the fact that hypotonically-induced ATP release in normal conditions was not accompanied with detectable permeation of PI, we conclude that ATP in A549 cells is released by a non-conductive pathway.

Immunological tolerance induced by liver grafting in the rat: splenic macrophages and T cells mediate distinct phases of immunosuppressive activity

In the rat combination DA into PVG, liver grafts are not rejected but induce donor-specific transplantation tolerance. We have examined the immunosuppressive properties of spleen cells from PVG recipients of DA liver grafts at various times post-grafting. The results indicate the development of two phases of cell-mediated suppressor activity, which appear to be mediated by separate spleen cell populations. Mitomycin-C-treated spleen cells taken from animals between 5 and 28 days post-grafting were able to suppress rat mixed lymphocyte reactions (MLRs). These 'early' suppressor cells were glass adherent and absent from populations purified by passage through nylon wool or G10 Sephadex columns. Suppression of MLR by purified glass adherent cells was not specific for either stimulator or responder haplotypes and was blocked by indomethacin. Nylon wool purified T cells were not suppressive at this time. Spleen cell suppressor activity declined to background levels after 35 days post-grafting. However, spleen cells from long-term surviving liver graft recipients (20 weeks or more) were again able to suppress MLR; the 'late' suppressor cells were nylon wool non-adherent and suppression was specific for the donor (DA) MHC type. We conclude that liver grafting in this combination generates early and late phases of suppression among spleen cells, that the early phase is produced by macrophages and mediated by prostaglandins and that the late phase is dependent on allospecific suppressor T cells.

Silver deposition on freeze-dried cells allows subcellular localization of cholesterol with imaging TOF-SIMS.

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used for characterization and subcellular localization of organic ions in leucocytes adhering to glass surfaces. The cells were fixed by freeze drying in 0.15 m ammonium formate buffer at pH 7.2-7.4. The freeze-dried cells were sputter-coated with silver, and the silver surface was analysed with imaging TOF-SIMS. TOF-SIMS spectra were recorded by scanning the primary ion beam over the analysis area and acquiring positive mass spectra of the ions leaving the surface. The relative brightness of each pixel within the analysis area reflects the signal intensity of a selected ion in that pixel. Data were collected separately at high mass resolution m/delta m > 7000 and at high lateral resolution (= 0.5 micro m). The images were analysed by principal component analysis (PCA). The glass-adhering cells showed a well defined attachment area with a diameter of up to 20 micro m, and an equally well defined cell body, containing the nucleus, with a diameter of 8-10 micro m. On the raw data images, the obtained cholesterol distributions were consistent with a higher cholesterol content of the cell membrane in the attachment area than in the cell body. Using PCA analysis, silver-cationized molecular cholesterol was found localized mainly in the attachment area of the cells. Cholesterol was also seen at higher concentration in circular spots of </= 1 micro m in diameter, probably representing caveolae.

AUGMENTATION OF SPECIFIC AND NON-SPECIFIC PROTECTION IN OREOCHROMIS NILOTICUS TREATED WITH SOME PREBIOTICS AND / OR BACTERIN

We experimentally infected Oreochromis niloticus (O.niloticus) fish with Hafnia alvei (H.alvei), the causative agent of fish haemorrhagic septicemia in order to compare the immunological responses to differ-ent treatments with glucan, alginate and / or in combination with H.alvei bacterin. Elevations in the non-specific defence mechanisms were noted after O. niloticus were bathed in glucan, alginate, and bacterin solutions or in solutions containing the glucan or alginate combined with a bacterin. Blood samples were collected in several times showed an elevation in neutrophil activity through the ability of the cells to stick to glass and produce oxidative radicals where detected by the nitroblue tetrazolium (NBT) assay which increased after treatments in all groups within 48 hours. The numbers of circulatory glass-adherent cells from fish given the alginate, glucan and / or bacterin by bath was significantly higher than at the begining of the experiments or the control levels. Elevated phagocytosis, assessed by increased uptake of glutaraldhyde-treated sheep red blood cells, also confirmed these kinetics. It is obvious that fish given alginate and glucan in combination with bacterin, the kinetics of the non-specific and specific immune responses were significantly remarkable. The most promising group of immunostimulant are the alginate and the glucan and they could play an important role as imm-unomodulators in fish farming and in the prevention of diseases in fish cultures. This paper will present data on such effects with reference mainly to prebiotics and how can induce a systemic effect.

Pronectin Promotes Calcium Signaling by Interferon-γ Human Neutrophils via G-Protein and Sphingosine Kinase-Dependent Mechanisms

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-gamma (IFN-gamma) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-gamma-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-gamma responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-gamma-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine 1-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-gamma partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-gamma. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-gamma induced calcium signals.

Natural killer activity in mice infected with rabies virus and submitted to P. acnes ( Propionibacterium acnes) as immunomodulator

The natural killer (NK) activity and lethality were evaluated in swiss mice experimentally infected with street rabies virus and submitted to immunomodulation by P. acnes (formerly Corynebacterium parvum). The infected animals were sacrificed at different times and spleen non-adherent cells were obtained through ficoll-hypaque gradient and depletion of glass-adherent cells. Immunosuppression was observed in rabies virus infected mice correlated with lower NK activity in clinically ill animals. Higher NK activity and percentual of survival were observed in the group submitted to P. acnes. The increased survival correlated with higher NK activity induced by P. acnes suggests a protective role of this natural barrier against rabies virus infection in mice.

Comparison of cytoskeletal structures in glass-adherent and polyethylene terephthalate (PET) membrane-adherent human pulmonary fibroblasts

In order to use microporous membranes as a culture substratum for later air/liquid exposures with human pulmonary fibroblasts, we were interested to find out whether the behaviour of cells grown on PET membranes differs from those cultured under conventional conditions. We therefore compared cytoskeletal structures of membrane-adherent and glass-adherent cells. There were no differences found between the cytoskeleton of cells grown on membranes or on glass. They attach equally to both of the substrates and develop the same cytoskeletal structures. We also investigated the proliferative activity of the fibroblasts on PET-cell culture inserts by determination of Pico Green intercalation into DNA and turnover of a tetrazolium salt (WST) by mitochondrial activity. In summary, the fibroblasts were found to show characteristic growth tendencies when cultivated on PET membranes.

Alpha-melanocyte stimulating hormone (α-MSH) and melanin-concentrating hormone (MCH) stimulate phagocytosis by head kidney leucocytes of rainbow trout (Oncorhynchus mykiss) in vitro

Alpha-melanocyte stimulating hormone (α-MSH) and melanin-concentrating hormone (MCH) are two peptides with antagonistic roles in the regulation of skin pigmentation in teleost fish. Both have also been implicated in the modulation of the stress response via the hypothalamo-pituitary-adrenal (HPA) axis in fish and other vertebrates. Alpha-MSH is also known to be a potent immunomodulatory peptide in mammals, while both hormones have been shown to influence the immune responses of troutin vitro. Head kidney phagocytes (macrophages and neutrophils) were exposed to α-MSH at concentrations of 0·05 to 10nMin vitro for 60min and the phagocytic activity of glass-adherent cells was assessed microscopically. At 1, 5 and 10nMα-MSH significantly increased the percentage of phagocytes that ingested heat-killed yeast cells. Percentage phagocytosis was also significantly increased when cells were exposed to 0·1, 1 and 10nM des-acetyl-α-MSH. Whenα-MSH and MCH were added to cells together, at concentrations of 1nM and 50nM respectively, the stimulatory effects of both hormones were diminished. The results suggest that these peptides may play an immunomodulatory role in the fish immune system.

Glucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout ( Oncorhynchus mykiss)

Elevations in the non-specific defence mechanisms were noted after trout were injected or bathed in glucan solutions or in solutions containing the glucan combined with a bacterin, Y. ruckeri O-antigen. Periodic non-lethal blood samples showed that neutrophil activity as evaluated by the ability of the cells to stick to glass and produce oxidative radicals as detected by the nitroblue tetrazolium (NBT) assay, rose after treatments within 2 days. Elevated phagocytosis, assessed by increased uptake of glutaraldehyde-treated sheep red blood cells, also confirmed these kinetics. The numbers of circulatory glass-adherent cells from fish given the glucans by injection or bath was twice the level of the sham-treated controls; likewise the phagocytic ratio also was two-fold higher. In following the kinetics of the non-specific defensive responses, the injection of the glucan caused an immediate, slight reduction of NBT staining cells and numbers of leukocytes before the 2–3 day rise; this was not obvious in the fish given the glucan by bath. The glucans could play an important role in the prevention of diseases in fish culture.

GM-CSF augments the immunosuppressive capacity of neonatal spleen cells in vitro

Addition of exogenous granulocyte-macrophage colony stimulating factor (GM-CSF) to cultures of adult murine spleen cells with sheep red blood cells (SRBC) results in an augmented plaque forming cell (PFC) response. The influence of GM-CSF on the ability of neonatal spleen cells to suppress the anti-SRBC plaque forming response of adult spleen cells was tested by adding GM-CSF to cultures of neonatal and adult spleen cells. The suppressive capacity of the neonatal spleen cells was augmented by exogenous GM-CSF. The augmented suppression of the neonatal spleen cells was dependent on a G-10 adherent population since the addition of GM-CSF to cultures containing G-10 passed neonatal spleen cells resulted in an augmented PFC response and not suppression. Neonatal splenic glass adherent cells were also capable of suppressing the response. Neonatal spleen cells or purified neonatal glass adherent spleen cells cultured in the presence of GM-CSF had markedly increased levels of PGE 2 in the culture supernatant. Neonatal spleen cells cultured with GM-CSF had increased numbers of morphologically identifiable macrophages after 48 hr of culture. BOth irradiation and G-10 passage of the neonatal spleen diminished the numbers of macrophages formed in response to GM-CSF, and both of these manipulations resulted in reversal of suppression in response to GM-CSF. Thus, the augmented suppressive capacity of neonatal spleen cells in response to GM-CSF is probably mediated by its ability to drive monocyte to macrophage differentiation as well as increase the suppressive capacity of the existing neonatal splenic macrophages by increasing their production of PGE 2.

Endocytosis via the scavenger- and the mannose-receptor in rainbow trout (Salmo gairdneri) pronephros is carried out by nonphagocytic cells

Endocytosis of modified human serum albumin (HSA) and mannose-terminated glycoproteins was studied in pronephros cells from rainbow trout (Salmo gairdneri). Blood clearance and tissue uptake of dinitrophenylated human serum albumin (DNP-HSA) was dependent on the number of DNP-groups conjugated to the albumin molecule. Uptake of DNP35-HSA in isolated pronephros cells was saturable. Pronephros cells also internalized the mannose-terminated glycoprotein invertase by a receptor-mediated process. DNP-HSA and invertase were recovered in the same cell fractions when pronephros cells containing in vivo endocytosed ligands were separated by density gradient centrifugation. The cells endocytosing these ligands were apparently not macrophages. The macrophages were recovered in cell fractions with higher densities. They were identified by their ability to adhere to glass and to carry out phagocytosis. Cultured macrophages did not endocytose chemically modified albumin (DNP-HSA and formaldehyde-treated albumin) and mannose-terminated glycoproteins (ovalbumin) in vitro. These ligands were not recovered in glass adherent pronephros cells after in vivo endocytosis of the proteins. The present data suggest that macrophages in rainbow trout pronephros possess neither the receptor for chemically modified albumin, the scavenger receptor, nor the mannose-specific receptor.

Leukocytes of rainbow trout ( Oncorhynchus mykiss) phonephros: Cell types producing superoxide anion

Fish phonephric and blood leukocytes yield a chemiluminescent response (CL) when stimulated appropriately. This response reflects the production of highly reactive oxygen derivatives which contribute to oxygen-dependent killing of targets such as pathogens and parasites. From suspensions of pronephric cells of the Rainbow trout, Oncorhynchus mykiss, we have obtained populations enriched for CL-positive cells. Four bands of cells were obtained using continuous gradients generated with 60% Percoll. The leukocytes of band II showed a very strong PMA-induced CL response, the magnitude of which was several times higher than that observed with equivalent numbers of cells form unseparated pronepheric cell suspensions. Cells present in other bands were not significantly chemiluminescent. Flow cytometric analysis showed that band II contained large granular cells and small granular cells. Cytochemical analysis showed that this subpopulation was greatly enriched with neutrophils. Many band II cells adhere to glass whereas few band III cells do so. The glass-adherent cells loose their CL potential after a few days in vitro, whereas the nonadherent cells retain their CL responsiveness for at least a week in vitro.

Development of surface membrane characteristics of "premedullary" macrophages in organ cultures of embryonic rat and hamster lungs.

A replicating population of non-monocyte-derived free cells appears in organ-cultured embryonic rat lungs, indistinguishable from alveolar macrophages by classical criteria such as ultrastructure, lysosomal enzyme cytochemistry, and phagocytic behavior. We demonstrate similar events in cultured embryonic hamster lungs and development of macrophage-associated properties on the plasmalemma of these cells in both species. Immunoperoxidase localizations were obtained using monoclonal antibodies against alveolar macrophage antigen (HAM1) in hamsters, and rat macrophage antigen (ED1) and leukocyte-common antigen (OX1) in rats. Fc and C3b receptors were identified in both species by immune rosetting. HAM1 staining, perinuclear in rare cells at explantation, gains definitive surface localization 3-4 days later as cells prepare to emerge through the pleura. ED1 and OX1 cytoplasmic staining first occurs after 24 hr, increases as macrophages multiply and congregate beneath the pleura, and translocates to the plasmalemma of emerged cells. Some glass-adherent cells from lung explants have Fc receptors. The proportion rises sharply for 24 hr and equals fully emerged cells (90-95%) by days 3-4. At first phagocytosis is slow to follow Fc receptor binding, but ingestion time decreases to 3-10 min as macrophages mature. A minority of emerged macrophages bind complement-opsonized erythrocytes, which are rarely taken up. These properties are shared by alveolar macrophages of adults.

Antibacterial activity of human natural killer cells.

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

Enhancement of in vitro cytotoxicity of mouse peritoneal exudate cells by flavone acetic acid (NSC 347512)

Flavone acetic acid (FAA), an antitumour agent currently undergoing clinical trials, was found to augment the tumoricidal activity of peritoneal exudate (PE) cells in vitro. Lysis of tumour targets was measured using a standard 18 h 51Cr release assay for activated macrophages. Lytic activity increased with increasing concentrations of FAA up to 100 μg/ml before reaching a plateau. At 80 μg/ml FAA, 3-fold fever PE cells were necessary to obtain the same level of activity as in control cultures without FAA. The lytic activity was mediated by Thy- 1 negative and glass-adherent cells in the PE population, and was inhibited by dexamethasone. The activity of PE cells against several different tumour targets (P815 mastocytoma, YAC- 1 lymphoma, P 388 lymphoma, and a Lewis lung carcinoma cell line) were all enhanced by FAA. The results show that FAA can enhance the lytic potential of peritoneal macrophages in vitro to kill a range of tumour cells.

Antimicrobial properties of Kupffer cells

To characterize the antimicrobial activities of Kupffer cells, I harvested macrophages from livers with a technique involving perfusion with collagenase and DNase. Ninety-nine percent of glass-adherent cells had typical macrophage morphology, 99% were esterase positive, and 60% phagocytosed opsonized zymosan when challenged with four particles per macrophage. Toxoplasma gondii multiplied within Kupffer cells from unmanipulated mice, but multiplication was intermediate between that observed in highly permissive peritoneal macrophages and highly activated macrophages. Intravenous injection of heat-killed Propionibacterium acnes, a stimulus known to activate macrophages in other compartments, resulted in a uniform, highly activated population of liver macrophages. Kupffer cells from P. acnes-injected mice were capable of generating reactive oxygen intermediates as shown by reduction of Nitro Blue Tetrazolium during phagocytosis of T. gondii or opsonized zymosan. In contrast, intravenous P. acnes injection did not activate spleen macrophages. Intravenous injection of P. acnes into athymic mice activated Kupffer cells, which suggested that T cells were not essential for this response. Kupffer cells were not activated in mice with latent Toxoplasma infection or during acute Giardia muris infection. Ordinarily, Kupffer cells became highly permissive for T. gondii during 48 h in culture, but inclusion of recombinant murine gamma interferon maintained their moderate inhibitory activity.

Suppression of liver regeneration resulting from intravenous injection of splenic glass adherent cells activated by poly I:C

Syngeneic spleen cells (SPCs) were treated with poly I:C in the presence of DEAE-dextran for 2 h in vitro for their activation (IC-SPCs). When these IC-SPCs were inoculated into 70 partially hepatectomized mice, liver regeneration of the mice was strongly suppressed. The extent of suppression was dependent upon the number of injected cells and upon the concentration of poly I:C for in vitro treatment. Among IC-SPCs, the suppressive activity was predominantly caused by splenic glass adherent cells pretreated with poly I:C (IC-SACs) than by pretreated splenic T cells (IC-T). IC-SACs additionally cultured without poly I:C for 24 h lost their suppressive activity; however, when they were cultured in the presence of indomethacin, they could retain their activity, interleukin 1 (IL 1) activity in culture supernatant was greater in the group cultured with indomethacin than without it. These results suggest that the suppressive activity of IC-SACs was not solely caused by the poly I:C carried into the mice with IC-SACs but by the activated IC-SACs themselves. The peak of liver cell mitosis in mice injected with IC-SACs was delayed 24 h as compared with non-injected groups, but IC-SACs-injected mice did not lose their regenerative potential. These findings demonstrate that the activated immune system may have some regulatory effects on liver regeneration.

Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｓｐ．に対するブリマクロファージの食菌作用に及ぼす抗体および補体のオプソニン効果

In order to know the defense mechanism of yellowtail Seriola quinqueradiata against Streptococcus sp, infection, phagocytic response of glass adherent macrophage-like cells isolated from normal fish was examined after one hour incubation at 25°C. Phagocytosis was observed in Giemsa stained smears under light microscope. Opsonic effects of specific antibody and com-plement were also investigated. Specific antibody increased the phagocytic activity and phagocytic index significantly. After addition of complement neither phagocytic activity nor phagocytic index changed significantly. In presence of specific antibody and complement, phagocytic activity and phagocytic index were significantly higher than in the control, and higher than in the previous tests. These results suggest that activation of the classical pathway of yellowtail complement by antigen-antibody reaction is an important function in opsonization of particulate antigen.

Phenotypic variation of Staphylococcus epidermidis slime production in vitro and in vivo.

Clinical studies performed by us and others have found an association between slime production and strains of coagulase-negative staphylococci that infect indwelling medical devices. By serial low-speed centrifugation of broth cultures we have isolated a stable, weakly adherent strain (RP62A-NA) from a strongly adherent, slime-producing, pathogenic strain of Staphylococcus epidermidis sensu stricto (RP62A, ATCC 35984). We obtained a second strain from RP62A-NA (RP62A-NAR) by serial subculture of glass-adherent cells of RP62A-NA. All three strains had the same pattern of biochemical reactions, antimicrobial susceptibilities, and plasmid analysis. Transmission electron micrograph sections stained with the mucopolysaccharide-specific stain alcian blue demonstrated that the adherent strains RP62A and RP62A-NAR were covered with an extracellular coat of polysaccharide-rich material. In contrast, the nonadherent RP62A-NA strain lacked this external coat. All three strains were used in a mouse model of foreign body infection and a rat model of catheter-induced infective endocarditis. The adherence characteristics of isolates of RP62A and RP62A-NA recovered from experimental animals were relatively stable, although we noted a slight but a significant increase in the adherence of RP62A-NA isolates recovered from the foreign body model. The adherence characteristics of RP62A-NAR isolates recovered from infected animals were variable; in general these isolates were less adherent than the laboratory strain of RP62A-NAR. In both models the 50% infective dose (calculated by the Reed and Muench method) was three times greater for the RP62A-NA strain than for the RP62A strain. The phenotypic expression of slime production is subject to both in vitro and in vivo variation and could play a role in the pathogenesis of foreign body infection.

Mechanisms of HLA-DR antigen expression in phytohemagglutinin-activated T cells in man. Requirement of T cell recognition of self HLA-DR antigen expressed on the surface of monocytes.

Signals required for expression of HLA-DR (DR) antigen in phytohemagglutinin (PHA)-activated human peripheral blood T cells were examined. T cells were purified by a four-step procedure, which included depletion of glass-adherent cells, 53% Percoll gradient centrifugation, nylon wool column passage, and treatment with mouse monoclonal antibodies directed to human HLA-DR antigen and Leu M1 antigen plus complement. Purified T cells responded poorly to PHA but with the combination stimuli of PHA and recombinant human interleukin 2 (rIL-2), resting T cells proliferated as well as T cells cultured with 10% monocytes and PHA. But well proliferated T cells in the absence of monocytes expressed very poor DR antigen after 7 to 8 days of culture. DR expression of T cells was restored by the addition of 10% monocytes. Allogeneic monocytes also helped proliferative responses of PHA-activated T cells but did not help the expression of DR antigen. These results suggested that signals required for T cell proliferation (PHA and rIL-2) were not sufficient for DR expression in this system and further monocytes were essentially required in a HLA-restricted manner. In the next experiment, we examined the role of membrane molecules in monocytes for transmission of signals that induce activated T cells to express DR antigen. Autologous monocytes were fixed with 1% paraformaldehyde and added to T cells in the presence of PHA and rIL-2. Fixed monocytes could help DR antigen expression of PHA-activated T cells as well as viable monocytes. But when fixed monocytes were pretreated with anti-HLA-DR monoclonal antibody, they could not help DR expression of T cells any longer. These results suggested that for the expression of DR antigen, PHA-activated T cells had to first recognize self DR antigen expressed on the surface of monocytes before proliferation occurred.