Applications of Bioinspired Platforms for Enhancing Immunomodulatory Function of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) have attracted scientific and medical interest due to their self-renewing properties, pluripotency, and paracrine function. However, one of the main limitations to the clinical application of MSCs is their loss of efficacy after transplantation in vivo. Various bioengineering technologies to provide stem cell niche-like conditions have the potential to overcome this limitation. Here, focusing on the stem cell niche microenvironment, studies to maximize the immunomodulatory potential of MSCs by controlling biomechanical stimuli, including shear stress, hydrostatic pressure, stretch, and biophysical cues, such as extracellular matrix mimetic substrates, are discussed. The application of biomechanical forces or biophysical cues to the stem cell microenvironment will be beneficial for enhancing the immunomodulatory function of MSCs during cultivation and overcoming the current limitations of MSC therapy.

The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.

Deoxypodophyllotoxin Induces a Th1 Response and Enhances the Antitumor Efficacy of a Dendritic Cell-based Vaccine

BACKGROUND: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-kappaB. METHODS: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. RESULTS: DPT promoted the activation of CD8+ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-gamma production and induction of cytotoxic T lymphocyte activity. CONCLUSION: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

The Effect of Caffeic Acid on Wound Healing in Skin-incised Mice

This study was carried out to investigate the wound healing effect of caffeic acid in skin-incised mice. Caffeic acid showed significant effects on anti-inflammatory activity and wound healing, such as myeloperoxidase activity, lipid peroxidation, phospholipase A2 activity and collagen-like polymer synthesis, in incised-wound tissue. On the other hand, it significantly stimulated collagen-like polymer synthesis in NIH 3T3 fibroblast cells, while inhibited both silica-induced reactive oxygen species generation and melittin-induced arachidonic acid release and PGE2 production in Raw 264.7 cells, and histamine release in RBL 2H3 cells stimulated by melittin or arachidonic acid. Therefore, caffeic acid appears to have a potent antioxidant and anti-inflammatory effect in cell culture system, which may be related to wound healing in skin-incised mice.

Inhibitory Effects of Valdecoxib on Sodium Currents in Sensory Neurons

BACKGROUND: Valdecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor. It is effective in the treatment of rheumatoid arthritis, osteoarthritis, primary dysmenorrhea, and postoperative pain. Two kinds of sodium currents, tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R), are expressed in the dorsal root ganglia (DRG). Both sodium currents are implicated in the formation of normal and abnormal pain. METHODS: The effects of valdecoxib on sodium currents in rat DRG neurons were investigated using the whole-cell variation of the patch-clamp technique. RESULTS: Valdecoxib suppressed two types of sodium currents in a dose-dependent manner, without altering the activation and inactivation kinetics of either current type. It shifted the activation voltage toward a depolarizing direction and the steady-state inactivation voltage toward a hyperpolarizing direction, and suppressed resting channels to similar extents in both types of sodium currents. Valdecoxib slowed the recovery of both sodium currents from inactivation, and suppressed them in a frequency-dependent manner. CONCLUSIONS: The results suggest that valdecoxib may produce analgesic effects through the inhibition of sodium currents in sensory neurons as well as COX-2.

The Effect of Dehydroepiandrosterone on Isoproterenol-induced Cardiomyopathy in Rats

We evaluated therapeutic and preventive properties of dehydroepiandrosterone (DHEA), a weak androgenic steroid, against isoproterenol-induced cardiomyopathy. The cardiomyopathy was induced by daily i.p. administration of isoproterenol to rats for five days. One group of rats were given with daily s.c. for 5 days during isoproterenol and the other group with daily s.c. DHEA for total 10 days, including 5 days before and during isoproterenol. The animals were killed after each treatment, and cardiac muscle failure was evaluated using histopathologic examination and biochemical indices. DHEA was found to reduce the damaged area and inhibit the elevation in the serum levels of glutamic oxaloacetic transaminase (SGOT), lactate dehydrogenase (LDH), skeletal muscle creatine kinase (CK) and heart creatine kinase (CK-MB) induced by isoproterenol. We also assayed widely used oxidative stress parameters, including thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase and glutathion peroxidase (GPx). DHEA decreased the escalated level of TBARS and enhanced the anti oxidant defense reaction with an increase in Mn-SOD and Cu/Zn-SOD. On the other hand, the treatment with DHEA did not affect catalase and GPx activity. The present study indicates that DHEA has a therapeutic and preventive effect against isoproterenol-induced cardiomyopathy and its effects may depend largely on the increase in SOD activity.

Protective Effect of Defibrotide on Splanchnic Injury following Ischemia and Reperfusion in Rats

A splanchic artery occlusion for 90 min followed by reperfusion of the mesenteric circulation resulted in a severe form of circulatory shock, characterized by endothelial dysfunction, severe hypotension, marked intestinal tissue injury, and a high mortality rate. The effect of defibrotide, a complex of single-stranded polydeoxyribonucleotides having antithrombotic effect, was investigated in a model of splanchnic artery occlusion (SAO) shock in urethane anesthetized rats. Occlusion of the superior mesenteric artery for 90 min produced a severe shock state, resulting in a fatal outcome within 120 min of reperfusion in many rats. Defibrotide (10 mg/kg body weight) 10 min prior to reperfusion significantly improved mean arterial blood pressure in comparison to vehicle treated rats (p<0.05). Defibrotide treatment also significantly attenuated in the increase of plasma amino nitrogen concentration, intestinal myeloperoxidase activity, intestinal lipid peroxidation, infiltration of neutrophils in intestine and thrombin induced adherence of neutrophils to superior mesentric artery segments. Superoxide anion and hydrogen peroxide production in 1 micrometer formylmethionylleucylphenylalanine (fMLP)-activated PMNs was inhibited by defibrotide in a dose-dependent fashion. Defibrotide effectively scavenged hydrogen peroxide, but not hydroxyl radical. Treatment of SAO rats with defibrotide inhibited tumor necrosis factor-alpha, and interleukin-1beta productions in blood in comparison with untreated rats. These results suggest that defibrotide partly provides beneficial effects by preserving endothelial function, attenuating neutrophil accumulation, and antioxidant in the ischemic reperfused splanchnic circulation.

The Improvement of Learning and Memory Ability of Normal Persons by BF-7

To investigate whether BF-7, extracted from Bombyx mori, improved learning and memory of ordinary people, Rey-Kim Memory Test was performed with placebo group (32 persons), 200 mg BF-7 treated group (33 persons) and 400 mg BF-7 treated group (34 persons). BF-7 enhanced significantly learning and memory function in a dose-dependent manner. To know how BF-7 plays such a positive role, we measured the concentration of acetylcholine in the brain from memory impairment animal model. Treatment of BF-7 significantly increased the concentration of acetylcholine. So, it is supposed that the protection of cholinergic neuron and/or keeping proper concentration of acetylcholine might be one of the mechanisms by which BF-7 improve brain function. So, our results suggest that the BF-7 is effective material for improving learning and memory ability.

Effect of Defibrotide on Rat Reflux Esophagitis

This study was aimed at evaluating the effect of defibrotide on the development of the surgically induced reflux esophagitis, on gastric secretion, lipid peroxidation, polymorphonuclear leukocytes (PMNs) accumulation, polymorphonuclear leukocytes adherence, superoxide anion and hydrogen peroxide production in PMNs, scavenge of hydroxyl radical and hydrogen peroxide, cytokine (interleukin-1beta, tumor necrosis factor-alpha) production in blood, and intracellular calcium mobilization in PMNs. Defibrotide did not inhibit the gastric secretion and not change the gastric pH. Treatment of esophagitis rats with defibrotide inhibited lipid peroxidation, and myeloperoxidase (MPO) in the esophagus in comparison with untreated rats. Defibrotide significantly decreased the PMN adherence to superior mesenteric artery endothelium in a dose-dependent manner. Superoxide anion and hydrogen peroxide production in 1microM formylmethionylleucylphenylalanine (fMLP) - or 0.1microgram/ml N-phorbol 12- myristate 13-acetate (PMA) -activated PMNs was inhibited by defibrotide in a dose-dependent fashion. Defibrotide effectively scavenged the hydrogen peroxide but did not scavenge the hydroxyl radical. Treatment of esophagitis rats with defibrotide inhibited interleukin-1beta production in the blood in comparison with untreated rats, but tumor necrosis factor-alpha production was not affected by defibrotide. The fMLP-induced elevation of intracellular calcium in PMNs was inhibited by defibrotide. The results of this study suggest that defibrotide may have partly beneficial protective effects against reflux esophagitis by the inhibition lipid peroxidation, PMNs accumulation, PMNs adherence to endothelium, reactive oxygen species production in PMNs, inflammatory cytokine production (i.e. interleukin-1beta), and intracellular calcium mobilization in PMNs in rats.

Effect of Amrinone, a Selective Inhibitor of Phosphodiesterase III, on PMNs-induced Cardiac Dysfunction in Ischemia/reperfusion

Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. Amrinone, a specific inhibitor of phosphodiesterase 3, has an antioxidant activity against PMNs. Therefore, we hypothesized that amrinone could attenuate PMNs-induced cardiac dysfunction by suppression of reactive oxygen species (ROS) produced fby PMNs. In the present study, we examined the effects of amrinone on isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. Amrinone at 25microM, given to hearts during the first 5 min of reperfusion, significantly improved coronary flow, left ventricular developed pressure (P< 0.001), and the maximal rate of development of left ventricular developed pressure (P< 0.001), compared with ischemic/reperfused hearts perfused with PMNs in the absence of amrinone. In addition, amrinone significantly reduced myeloperoxidase activity by 50.8%, indicating decreased PMNs infiltration (p< 0.001). Superoxide radical and hydrogen peroxide production were also significantly reduced in fMLP- and PMA-stimulated PMNs pretreated with amrinone. Hydroxyl radical was scavenged by amrinone. fMLP-induced elevation of [Ca2+]i was also inhibited by amrinone. These results provide evidence that amrinone can significantly attenuate PMN-induced cardiac contractile dysfunction in the ischemic/ reperfused rat heart via attenuation of PMNs infiltration into the myocardium and suppression of ROS release by PMNs.

Dietary Ascorbate Supplementation Reduces Oxidative Tissue Damage and Expression of iNOS in the Kidney of Streptozotocin Induced Diabetic Rats

Reactive oxygen species (ROS) have been suggested to be contributory factors in complications of diabetes mellitus. In the present study, we investigated the generation of superoxide, the lipid peroxide level measured as thiobarbituric acid reactive substances, the vasorelaxation of isolated thoracic aorta and the iNOS expression in kidney of streptozotocin induced diabetic rats. Sprague Dawley rats were divided into four groups: control, ascorbate (400 mg/kg rat weight daily in drinking water), diabetic (single dose of 50 mg of STZ/kg i.p.) and diabetic simultaneously fed with ascorbate for 12 wk. Rats in groups were studied at tri-weekly intervals (0 to 12 wk). Diabetic rats were evaluated periodically with changes of plasma glucose levels and body weight. The ascorbate supplimentation attenuated the development of hyperglycemia and weight loss induced by STZ injection in rats. In the present experimental condition, the ascorbate supplimentation had no significant effect on plasma glucose levels and changes in body weight of normal rate. The superoxide generation, formation of thiobarbituric acid reactive substance and iNOS expression in kidney were significantly increased in STZ-treated rats that were decreased by ascorbate supplimentation. The ascorbate supplimentation had no effect on vasorelaxation of isolated thoracic aorta. These results indicate that ascorbate supplimentation may exert an inhibitory effect on STZ-induced oxidative tissue damage through protection of pancreatic islet cells by scavanging reactive oxygen species. The ascorbate supplimentation may possibly attenuate the renal complication of diabetes mellitus.

Protective effect of rutin on splanchnic injury following ischemia and reperfusion in rats

A splanchic artery occlusion for 90 min followed by reperfusion of the mesenteric circulation resulted in a severe form of circulatory shock characterized by endothelial dysfunction, severe hypotension, marked intestinal tissue injury, and a high mortality rate. The effect of rutin, a flavonoid having antiprostanoid, anti-inflammatory, antithrombotic, antioxidant effect, were investigated in a model of splanchnic artery occlusion (SAO) shock in urethane anesthetized rats. Occlusion of the superior mesenteric artery for 90 min produced a severe shock state resulted in a fatal outcome within 120 min of reperfusion in many rats. Rutin was given as a bolus (1.28 mg/kg) 10 min prior to reperfusion. Administration of rutin significantly improved mean arterial blood pressure in comparison to vehicle treated rats (p<0.05). Rutin treatment also resulted in a significant attenuation in the increase in plasma amino nitrogen concentration, intestinal myeloperoxidase activity, intestinal lipid peroxidation, infiltration of neutrophils in intestine and thrombin induced adherence of neutrophils to superior mesentric artery segments. These results suggest that rutin provides beneficial effects in part by preserving endothelial function and attenuating neutrophil accumulation in the ischemic reperfused splanchnic circulation.

Effects of Fluoxetine on Sodium Currents in Rat Sensory Neurons

BACKGROUND: Fluoxetine, a widely used antidepressant drug, has been described as a selective serotonin reuptake inhibitor. In addition to its antidepressant action it has been demonstrated to be effective in alleviating pain associated with various diseases. Dorsal root ganglion (DRG) neurons are primary sensory neurons and transmit peripheral information to central nervous system. Two types of sodium channels are expressed in DRG neurons based on their sensitivity to tetrodotoxin. They are involved in the generation and conduction of nociception. The effects of fluoxetine on sodium currents in DRG neurons were examined to elucidate the analgesic mechanism of the drug. METHODS: DRG neurons wereacutely dissociated from rats (2~6 days postnatal) by enzymatic digestion. The whole-cell configuration of patch clamp technique was used to record tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents. RESULTS: Fluoxetine inhibited TTX-S and TTX-R sodium currents with Kd values of 60 microM and 59 microM, respectively, at the holding potential of -80 mV. For both types of sodium channels the steady-state inactivation curves were shifted in the hyperpolarizing direction and the conductance-voltage relationship curves were shifted in the depolarizing direction by fluoxetine. These effects combined together would greatly reduce the neuronal excitability. CONCLUSIONS: The blockade of sodium currents in sensory neurons is considered as a possible mechanism for the analgesic action of fluoxetine.

Myocardial protection by recombinant soluble P-selectin glycoprotein ligand-1: Suppression of neutrophil and platelet interaction following ischemia and reperfusion

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Additional control rat hearts were perfused with 75 X 106 PMNs, with 75 X 106 platelets, or with 75 X 106 PMNs+75 X 106 platelets over a five minute perfusion followed by a 75 min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 5 ~ 10% (p < 0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50 to 60% in all measurements of cardiac function (p < 0.01). These dual cell perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiaodynamic effects as well as PMN accumulation and P-selectin expression were markedly attenuated by a recombinant soluble PSGL-1 which inhibits selectin mediated cell adhesion. These results provide evidence that platelets and PMNs act synergistically in provoking post-reperfusion cardiac dysfunction, and that this may be largely due to cell to cell interactions mediated by P-selectin. These results also demonstrate that a recombinant soluble PSGL-1 reduces myocardial reperfusion injury by platelet and PMNs interaction.

Effect of C1 esterase inhibitor on the cardiac dysfunction following ischemia and reperfusion in the isolated perfused rat heart

Complement-mediated neutrophil activation has been hypothesized to be an important mechanism of reperfusion injury. It has been proposed that C1 esterase inhibitor (C1 INH) may prevent the complement-dependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium. Therefore, The effect of C1 INH was examined in neutrophil dependent isolated perfused rat heart model of ischemia (I) (20 min) and reperfusion (R) (45 min). Administration of C1 INH (5 mg/Kg) to I/R hearts in the presence of PMNs (100 X 106) and homologous plasma improved coronary flow and preserved cardiac contractile function (p<0.001) in comparison to those I/R hearts receiving only vehicle. In addition, C1 INH significantly (p<0.001) reduced PMN accumulation in the ischemic myocardium as evidenced by an attenuation in myeloperoxidase activity. These findings demonstrate the C1 INH is a potent and effective cardioprotective agent inhibits leukocyte-endothelial interaction and preserves cardiac contractile function and coronary perfusion following myocardial ischemia and reperfusion.

Enhancement of cyclosporine-induced oxidative damage of kidney mitochondria by iron

The present study investigated the stimulatory effects of iron (or ascorbate) on cyclosporine-induced kidney mitochondrial damage. Damaging effect of 50 muM cyclosporine plus 20 muM Fe2+ on mitochondrial lipids and proteins of rat kidney and hyaluronic acid was greater than the summation of oxidizing action of each compound alone, except sulfhydryl oxidation. Cyclosporine and 100 muM ascorbate showed an enhanced damaging effect on lipids but not on proteins. The peroxidative action of cyclosporine on lipids was enhanced with increasing concentrations of Fe2+. Ferric ion (20 muM) also interacted with cyclosporine to stimulate lipid peroxidation. Damaging action of cyclosporine on mitochondrial lipids was enhanced by ascorbate (100 muM and 1 mM). Iron chelators, DTPA and EDTA, attenuated carbonyl formation induced by cyclosporine plus ascorbate. Cyclosporine (100 muM) and 50 muM Fe2+ (or 100 muM ascorbate) synergistically stimulated degradation of 2- alpha deoxyribose. Cyclosporine (1 to 100 muM) reduced ferric ion in a dose dependent manner, which is much less than ascorbate action. Addition of Fe2+ caused a change in absorbance spectrum of cyclosporine in 230~350 nm of wavelengths. The results show that cyclosporine plus iron (or ascorbate) exerts an enhanced damaging effect on kidney mitochondria. Iron and ascorbate appear to promote the nephrotoxicity induced by cyclosporine.

Immunohistochemical Analysis of MHC Class 2 (HLA-DR / DP), ICAM-1, CD68(+) Macrophage Expression in Gastric Adenocarcinoma / 대한암학회지

PURPOSE: Gastric adenocarcinoma is the most common malignant tumor in Korea and immunochemotherapy can be alternative method of the treatment for it. So we evaluated several immunologic markers, Major Histocomatibility (MHC) Antigen and Intercellular Adhesion Molecule (ICAM)-1 which play an important roles in cellular immune response of the host to the tumar cells, HLA-DR/DP antigens, one of the MHC class II which is expressed in various conditions, CD 68 antigen which are also important factor in immune response to the tumor cells. MATERIALS AND METHODS: We compared the expression of MHC class II (HLA-DR/DP) antigens, ICAM-1 and the number of tumor-infiltrating macrophages presenting CD68 antigen in formalin-fixed paraffin-embedded tissue sections of 95 gastric adenocarcinomas using immunohistochemistry. In addition to analyze the relationship between expression of these antigens in gastric adenocarcinoma, histolopathologic findings such as tumor invasion, regional lymph node metastasis and histologic differentiation are evaluated. RESULTS: The rate of HLA-DR/DP expression was 60% and strongly associated with tumor differentiation, invasion and regional lymph node metastasis. ICAM-1 was expressed in 15% and slightly increased in well-differentiated carcinoma. The lack of expression of ICAM-1 was observed in high invasive tumor (T 4). CD 68(+) macrophages counts were significantly increased in around the tumor cells, compared to normal epithelia. HLA-DR/DP expression and infiltrating CD 68(+) macrophage numbers were significantly associated (p<0.05), but there was no correlationship between ICAM-1 and CD 68(+) macrophage numbers. CONCLUSION: It was considered that enhanced expression of HLA-DR/DP antigens, ICAM-1 and CD68(+) macrophages in gastric adenocarcinomas may be an immunophenotypic deviation. HLA-DR/DP and CD68(+) macrophages infiltration showed correlationship with tumor invasion and regional lymph node metastasis, that they may be used as a prognostic factor of the tumor growth.

Comparison of regulatory action of cAMP and cGMP on the activation of neutrophil responses

The regulatory role of cyclic nucleotides in the expression of neutrophil responses has been examined. fMLP-stimulated superoxide production in neutrophils was inhibited by dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), histamine, adenosine + theophylline, cAMP elevating agents, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and sodium nitroprusside, cGMP elevating agents. Staurosporine, a protein kinase C inhibitor, genistein, a protein tyrosine kinase inhibitor and chlorpromazine, a calmodulin inhibitor, inhibited superoxide production by fMLP, but they did not further affect the action of DBcAMP on the stimulatory action of fMLP. DBcAMP, histamine, adenosine + theophylline and genistein inhibited myeloperoxidease release evoked by fMLP, whereas BrcGMP, sodium nitroprusside and staurosporine did not affect it. The elevation of (Ca2+)-i evoked by fMLP was inhibited by genistein and chlorpromazine but was not affected by staurosporine. DBcAMP exerted little effect on the initial peak in (Ca2+)-i response to fMLP but effectively inhibited the sustained rise. On the other hand, BrcGMP significantly inhibited both phases. fMLP-induced Mn-2+ influx was inhibited by either DBcAMP or BrcGMP. These results suggest that fMLP-stimulated neutrophil responses may be regulated by cAMP more than cGMP. cAMP and cGMP appear not affect stimulated responses by direct protein kinase C activation. Their regulatory action on the stimulated neutrophil responses may be not influenced by other activation processes.