Effect of physical stress on sensitivity of lymphocytes to inhibition by prostaglandin E2

Physical stress is associated with depressed cellular immune function. We have found that lymphocytes from subjects undergoing either of 2 stressful events, cardiac surgery or childbirth, are more sensitive to inhibition by PGE2. For example, the concentration of PGE2 required for 50% inhibition of 3H-thymidine incorporation (ID50) into phytohemagglutinin-stimulated lymphocytes from patients undergoing cardiac surgery went from 1.5 X 10(-8) M on the day before surgery to 3 X 10(-9) M on the day after surgery. This increase in sensitivity to PGE2 was accompanied by a significantly decreased lymphocyte proliferative response (27 to 68% of control, depending on mitogen dose) and a 50% increase in the percentage of E rosette-positive cells with receptors for the Fc portion of IgG. The increased sensitivity to PGE and the depressed mitogen responses returned to preoperative values by day 10. The depressed mitogen responses of the postoperative patients were completely restored to normal by removal of glass-adherent cells before culture. In addition, the responses of the postoperative patients and the women in labor were partially restored by the addition of indomethacin, a prostaglandin synthetase inhibitor, to the cultures. Thus it would appear that physical stress causes lymphocytes to become more sensitive to prostaglandin E2, and the increased sensitivity to inhibition by this immunomodulator is responsible in part for the depressed cellular immune function after physical stress.     

Supernatant derived from a human hepatocellular carcinoma cell line (PLC/PRF/5) activates a population of T-suppressor cells

Harvest fluid derived from a primary hepatocellular carcinoma cell line (PLC/PRF/5) inhibited the incorporation of 3H-thymidine into PHA-activated human lymphocytes. A similar effect was observed when lymphocytes were pre-incubated with the tumour supernatant and washed prior to mitogen activation. Not only did the tumour supernatant inhibit 3H-thymidine incorporation by mitogen-activated lymphocytes, but it also inhibited production of the lymphokine leucocyte inhibitory factor (LIF). In experiments designed to establish whether a component of the tumour harvest fluid was activating a population of suppressor cells, normal mononuclear (MN) cells were treated with the PLC/PRF/5 or embryonic fibroblast supernatant for 48 h, after which they were washed and added to normal mitogen-activated lymphocyte cultures. Only cells pretreated with the PLC/PRF/5 supernatant suppressed mitogenesis. The cell responsible for the suppressor effect was a T cell, which after a further 24 h in culture liberated a suppressor factor responsible for inhibiting lymphocyte function. Although the nature of the factor/s in the PLC/PRF/5 supernatant responsible for activation of the T-suppressor cell population is unknown, it is suggested that this mechanism may be important in protecting the tumour from the immune response.     

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.     

Regulation by prostaglandin E2 of interleukin release by T lymphocytes in mucosa

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E₂ in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E₂ is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E₂ modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E₂'s regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E₂concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E₂, when present in as low a concentration as 10⁻¹⁰M. PGE₂ also inhibited(by ≥ 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E₂ dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [³H] thymidine uptake studies suggested this was not due to a prostaglandin E²-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E², e.g., high concentrations (10⁻⁶ M) of prostaglandin E² inhibited IL-3 release by only 41%. We conclude that prostaglandin E² in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there. © 1996 Wiley-Liss, Inc.     

Migration inhibitory factor (MIF) and proliferative responses by human lymphocyte subpopulations separated by sheep erythrocyte rosette formation.

Human peripheral blood lymphocytes were separated by a combination of rosette formation with sheep erythrocytes and differential density centrifugation into subpopulations of rosette positive (T-enriched) cells and rosette negative (T depleted) cells. These were then tested in vitro for the production of macrophage migration inhibitory factor (MIF) and for incorporation of ³H-thymidine in response to specific antigens. Both T enriched and T depleted cell populations produced MIF but only T enriched cells exhibited significant antigen-induced ³H-thymidine incorporation. These findings using a T cell surface marker as the basis for cell separation, a technique which should not alter the B cell surface, confirm an earlier report in which human cells were separated on the basis of surface immunoglobulin, a B cell marker.     

Cytotoxicity of resident and lymphokine-activated mouse peritoneal macrophage against Trichomonas vaginalis

This study was aimed to observe the direct and lymphokine-activated cell mediated cytotoxic effects against Trichomonas vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured as release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. A 0.1 ml suspension of labeled protozoa (2 x 10(5)/ml) was placed in each well, followed by 0.1 ml of a suspension containing increasing numbers of peritoneal cells. After a 24 hr incubation at 37 degrees C, 0.1 ml of the supernatant was collected and counted in liquid scintillation counter. Mouse peritoneal macrophages had appreciable level of spontaneous cytotoxicity against T. vaginalis at the effector to target cell ratios from 5:1 to 50:1. Treatment of macrophages with lymphokine, produced by PHA-stimulated spleen cells, increased the cytotoxicity in comparison with resident macrophages against T. vaginalis. The degree of macrophage activation for the killing was not dependent upon the lymphokine concentration. Peritoneal cells adherent to plastic displayed significant levels of cytotoxicity against T. vaginalis. This study indicates that mouse peritoneal macrophages are spontaneously cytotoxic for T. vaginalis and lymphokine increases the cytotoxicity by activating macrophages to kill T. vaginalis.     

Immune response in mice exposed to low-dose X-rays

The present paper was conducted to evaluate the immunological effect of low dose gamma-irradiation. The splenocytes of mice (C57BL/6N), 24 hours after the irradiation from 0.087 to 0.87 Gy, were incubated with mitogens of T or B lymphocytes, allo-antigen (splenic cells of BALB/c mice) (MLC) or sheep red blood cells (SRBC) 10 days after immunization with the SRBC in vivo, and then their incorporations of 3H-thymidine were measured. On the other hand, this incorporation in the presence of T-cell growth factor (IL-2) in vitro and a drug of AET in vivo was investigated to examine their radioprotective effects. The dose-response relation, i.e. decrease of 3H-thymidine incorporation in function of increase of the irradiation dose, was demonstrated in these immunological examinations except at the dose of 0.087 Gy. More, their incorporation was remarkably promoted by the administration of the T cell growth factor and the drug, therefore, these substances represent the radioprotective effect.     

Independent regulation of prostaglandin production and the stress response in human fibroblasts.

The stress, or heat shock response of eukaryotic cells is characterized by dramatic changes in the metabolism of responding cells, most notably the increased synthesis of a group of proteins known as heat shock proteins. In this study, we examined the relationship of prostaglandin synthesis/release to the stress response. Stress protein synthesis was induced with sodium arsenite, and prostaglandin E2 and prostacyclin (measured as 6-keto PGF1 alpha) levels were determined by enzyme immunoassay. The stress response was monitored by the incorporation of [35S]methionine and separation of protein by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prostaglandin synthesis and the stress response were both induced by sodium arsenite. However, aspirin, a cyclooxygenase inhibitor, inhibited arsenite-induced prostaglandin synthesis but did not inhibit stress protein synthesis. Conversely, the calcium ionophore A23187 also stimulated prostaglandin synthesis, but did not induce the stress response. The results of this study indicate that sodium arsenite, a stress response inducer, stimulates prostaglandin production, but this appears to be a correlative rather than causative occurrence in the stress response. Determination of the cytotoxicity of arsenite indicated a high correlation of stimulation of prostaglandin release with cytotoxicity.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 × DDD)F₁ mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and ³H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much ³H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.     

Enrichment of RAW264.7 macrophages with essential 18-carbon fatty acids affects both respiratory burst and production of immune modulating cytokines

Macrophages play a vital role in the innate immune system. Thereby, production of both reactive oxygen intermediates and immune modulating cytokines is crucial for successful pathogen defense. Fatty acids may interfere with immune response in several ways. In this study, we investigated the influence of essential polyunsaturated fatty acids (PUFA) on key macrophage functions. RAW264.7 macrophages were cultured in a medium supplemented with 2 or 15 μmol/L of the n-6 PUFA linoleic acid (LA) or of the n-3 PUFA α-linolenic acid (LNA), respectively. Cells were tested for incorporation of fatty acids as well as NADPH oxidase activity. Furthermore, supernatants were collected for detection of NO and cytokine release (TNF-α, IL-6, IL-10). Exposure of RAW264.7 macrophages to LA or LNA resulted in incorporation of these fatty acids and their derivatives. Thereby, supplementation with both LA and LNA caused a significant increase in NADPH oxidase activity. In contrast, synthesis of NO was not affected by PUFA supplementation. Moreover, distinct effects could be seen in the release of immune modulating cytokines. Due to enhancement of NADPH oxidase activity, PUFA presumably promote the killing of pathogens crucial in host defense. In addition, the unsaturated fatty acids tested in our study were shown to modulate cytokine release by the macrophages, thus driving immune response into an anti-inflammatory direction. Of note, distinct differences between the n-6 PUFA LA and the n-3 PUFA LNA underline the impact of PUFA family on immune response.     

Lymphokine maintains macrophage activation for tumor cell killing by interfering with the negative regulatory effect of prostaglandin E2

Mouse resident peritoneal macrophages activated with bacterial lipopolysaccharide (LPS) rapidly lost their ability to kill tumor cells in vitro. Such loss of killing has previously been attributed to the effects of prostaglandin E (PGE) produced by the LPS-stimulated macrophages. Macrophages exposed in the current study to both LPS and partially purified lymphokine did not lose cytolytic activity, in spite of the fact that these cells produced undiminished amounts of PGE, compared to controls. Cytolytic activity was shown to be retained under these conditions because lymphokine decreased the sensitivity of activated macrophages to the negative regulatory effects of PGE. The mechanism responsible for the lymphokine effect is not known; however, generalized inhibition of macrophage responsiveness to the hormone does not appear to be involved because lymphokine did not reduce the cyclic AMP response of macrophages, measured on a whole cell basis, after they were exposed to PGE.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Prostaglandin E inhibition of mitogen stimulation in patients with multiple sclerosis

Kirbey et al have reported that leukocyte function from patients with multiple sclerosis is not suppressed by PGE2, as are normal leukocytes. We examined the ability of PGE2 (0.01-0.5 microgram/ml) to suppress Phytohemagglutinin induced 3H-thymidine incorporation in peripheral blood lymphocytes from multiple sclerosis patients and normals. There was no difference in sensitivity between the two groups. There was also no difference in activity of the prostaglandin producing suppressor cell between the multiple sclerosis patients and controls.     

1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells.

1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.     

Increase in pituitary adenosine 3′:5′-cyclic monophosphate content and potentiation of growth-hormone release from heifer anterior pituitary slices incubated in the presence of 3-isobutyl-1-methylxanthine

The release of growth hormone from heifer anterior pituitary slices and the cyclic AMP content of the slices were increased by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, both increases being related to inhibitor concentration over the range 0.1-1.0mm. Neither Ba(2+) (6.9 or 2.3mm), K(+) (72mm), nor p-chloromercuribenzoate (20mum) had any effect on pituitary cyclic AMP content over a 20min period. 3-Isobutyl-1-methylxanthine potentiated the release of growth hormone in response to Ba(2+) (2.3mm) and K(+) (24mm), but the degree of potentiation did not depend on inhibitor concentration in the same way as did tissue cyclic AMP content. 3-Isobutyl-1-methylxanthine decreased the concentration of K(+) required to give maximum stimulation of growth-hormone release, but did not significantly increase the maximum response to Ba(2+). Growth-hormone release in the presence of prostaglandin E(2) (1mum) was increased by 3-isobutyl-1-methylxanthine and was inhibited by the prostaglandin antagonist, 7-oxa-13-prostynoic acid, although this antagonist increased the pituitary cyclic AMP concentration and potentiated the prostaglandin E(2)-induced rise in cyclic AMP content. The stimulation of growth-hormone release by p-chloromercuribenzoate was not potentiated by 3-isobutyl-1-methylxanthine. The data suggest that Ba(+) and K(+) act at the same point in the secretory process as 3-isobutyl-1-methylxanthine, although by a different mechanism, and that p-chloromercuribenzoate has a different point of action.     

Platelet-derived growth factor stimulates phospholipase C-gamma 1, extracellular signal-regulated kinase, and arachidonic acid release in rat myometrial cells: contribution to cyclic 3',5'-adenosine monophosphate production and effect on cell proliferation

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-beta receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-gamma 1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI(2)). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA(2) inhibitor, and in the absence of Ca(2+). U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI(2) biosynthesis. PDGF-BB also increased cell proliferation and [(3)H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.     

Clonal analysis of mechanisms of murine T helper cell collaboration with effector cells of macrophage lineage

The molecular basis and genetic restrictions of collaboration between Th cells and macrophages (Mo) and the numbers of types of collaboration in the Ag-specific cellular immune response were analyzed. Using the response of cloned Ag-specific T cells we examined the mechanisms of induction of the macrophage procoagulant response. Two generic types of collaboration were identified. One was mediated by the lymphokine monocyte procoagulant inducing factor (MPIF) and the second mechanism was by apparent contact collaboration. The lymphokine MPIF was produced by T cells and cloned CD4+ T cells after specific Ag stimulation. Cloned CD8+ cells, most of which also exhibited cytolytic activity, produced little MPIF. There was no evident restriction of the response of Mo of different MHC or background genes. In the second collaborative pathway a subset of CD4+ cloned Th cells were able to directly collaborate by an apparent contact mechanism with Mo for the procoagulant response. There was no correlation of this latter capacity with MPIF production. In addition abrogation of protein synthesis and lymphokine production by Ag-driven clones did not abrogate the direct cell association type of collaboration. Both forms of collaboration were equally efficient across MHC incompatibility barriers and different genetic background. We conclude that there are two independent and parallel Th:Mo collaborative pathways for Ag-driven responses in this limb of the cellular immune response, i.e., a MPIF lymphokine pathway and a contact pathway, and that there are quantitative and qualitative clonal differences in the use of these two pathways. We suggest that the existence of multiple parallel pathways for cellular collaboration may occur more widely in the Th:Mo limb of the immune response in respect to other Mo effector molecules and should be explored to understand the orchestration of this limb of the immune response.     

The effects of a macrophage-derived cytotoxin on the growth and metabolism of target cells.

Peritoneal macrophages obtained from Sarcoma I (SaI)-immune CS7BL/6 mice release a heat-labile cytotoxin, specific macrophage cytotoxin (SMC), following a two hour interaction with appropriate target cells. Specific macrophage cytotoxin specifically inhibits A/Jax spleen cells from mitogenically responding to concanavalin A, whereas syngeneic CS7BL/6 spleen cells are unaffected. Treatment of target cells with SMC results in early alterations in RNA and DNA metabolism. The uptake and incorporation of ³H-uridine was found to be initially elevated while intracellular levels of ³H-thymidine became markedly reduced. Furthermore, the cytotoxic action of SMC was found to be rapidly accelerated and amplified by low levels of actinomycin-D.     

Prostaglandin synthesis and release by subpopulations of rat alveolar macrophages

Alveolar macrophages are the primary phagocytic cell of lung, but are also capable of a variety of other functions, which include initiating or modulating inflammatory and immune responses through the production of soluble mediators. One such group of mediators is the eicosanoids. Further, recent data indicate that alveolar macrophages are not functionally homogeneous, but are heterogeneous with several subpopulations that differ both morphologically and functionally. Considering the apparent importance of prostaglandin synthesis and release in inflammatory and immune responses, the current study was undertaken to determine whether alveolar macrophage subpopulations differ in their ability to synthesize and release prostaglandin (PG) E, PGI2, and thromboxane A2 after stimulation by calcium ionophore A23187, zymosan, or aggregated IgG. Alveolar macrophages were harvested by bronchoalveolar lavage and were separated into 18 density-defined fractions. Density-defined alveolar macrophages (DD-AM) showed marked heterogeneity in prostaglandin synthesis and release. Maximal PGE synthesis and release was seen as a single peak after calcium ionophore A23187 and zymosan stimulation. In contrast, two peaks in PGE synthesis were seen after aggregated IgG stimulation. PGI2 synthesis was seen as a single peak generated by different DD-AM after calcium ionophore A23187 and zymosan. In contrast, aggregated IgG stimulation of subpopulations exhibited uniform synthesis and release of PGI2. Thromboxane A2 synthesis and release was maximal from a broad range of various DD-AM after calcium ionophore A23187, zymosan, and aggregated IgG stimulation. The results demonstrate that DD-AM are heterogeneous in ability to synthesize and release prostaglandins which is dependent on the stimuli. Therefore, specific subpopulations of alveolar macrophages may be central to the control of the pulmonary inflammatory response through specific eicosanoid synthesis and release.