Capodicasa et al. (9) investigated the cytotoxic effects of omeprazole (OME) and OME-HCl (hydrochloric acid) on PMNs. PMNs were isolated from 15 healthy donor blood samples incubated with OME (unacidified or acidified) at a concentration range of 1 × 10⁻⁴ to 2.5 × 10⁻⁵ for 1–14 h. After 10 h of incubation, both activated and prodrug OME induced apoptosis of neutrophils. The prodrug OME-induced apoptosis was reduced via caspase-3 inhibition, whereas activated OME-induced apoptosis was reduced via both caspase-3 and caspase-8. Pantoprazole (PAN) is also capable of inducing caspase-3-dependent cell death by enhancing mitochondrial stress because of proteasomal inhibition and unfolded protein accumulation (10).

Wandall (11) used PMNs from healthy donors exposed for 30 min to unacidified or HCl-acidified OME at concentrations from 1 × 10⁻³ to 5 × 10⁻⁵ to assess chemotaxis, O₂ ⁻ generation, and degranulation. Preincubation of PMNs with unacidified or acidified OME reduced chemotaxis and degranulation in a dose-dependent manner compared to untreated PMNs. However, only acidified OME was shown to inhibit the generation of O₂ ⁻ with a half-maximal inhibitory concentration (IC₅₀) of 2.5 × 10⁻⁶. Suzuki et al. (12) used PMNs from 10 healthy human subjects given OME 20 mg/day for 7 days to measure ROS production. A dose-dependent reduction in oxidative radicals following stimulation with both N-formylmethionine-leucyl-phenylalanine (fMLP) and zymosan was observed. The intralysosomal pH also demonstrated a dose-dependent increase following OME exposure and fMLP or zymosan stimulation. Capodicasa et al. (13) used PMNs from 26 healthy volunteers to measure the effects of lansoprazole (LAN) on chemotaxis and O₂ ⁻ generation. Following a 30-min exposure to non-acidified LAN concentrations from 50 to 1,000 μM, a dose-dependent inhibition in chemotactic movement and superoxide production was observed. Zedtwitz-Liebenstein et al. (14) used blood samples from 10 healthy subjects at baseline and then 4 h after receiving OME 40 mg. Neutrophils were analyzed for their capacity to phagocytose Escherichia coli and the generation of ROS and intracellular calcium concentrations were measured. Phagocytosis of E. coli was not impaired after OME treatment, but the destruction of phagocytosed bacteria was significantly reduced. While basal extracellular ROS production was unaffected by OME, intra- and extracellular ROS production was reduced after stimulation, and cytosolic calcium increased. Nakagawa et al. (15) used RAW264.7 murine MΦs treated with LAN 0–400 µM for 2 h before a 12-h incubation with lipopolysaccharide (LPS) 1 µg/mL to assess P-ATPase mRNA, intracellular and extracellular ROS, NADPH oxidase activity, cyclooxygenase-2 (COX-2), inducible nitrous oxide synthase (iNOS), and prostaglandin E₂ (PGE₂) expression. Nitrous oxide (NO) and PGE₂ production was inhibited in a dose-dependent manner. Decreased NO production was associated with NADPH oxidase (NOX) inhibition suggesting a link between NO production and ROS, a reactive species produced by NOX. LAN inhibited ROS production following stimulation with LPS in a NADPH-oxidase-dependent manner. Since LAN inhibited the stimulation of NO induced by LPS, but not NO production by xanthine plus xanthine-oxidase (X/XO), a NADPH-oxidase-independent pathway, the authors conclude that LAN directly inhibits NADPH oxidase. Taken together, these studies demonstrate the OME inhibitory effects on the generation of ROS. ROS downregulation was observed with an increase in intracellular Ca²⁺. The mobilization of intracellular calcium plays an important role in ROS generation within the lysosome (16) as well as phagocytosis as it drives the polymerization of actin and the ingestion of extracellular components (17). These findings suggest that OME does not exert its effects through inhibition of Ca²⁺ signaling and Ca²⁺ mobilization, which conflicts with the results observed in Handa et al. (18) and Martins De Oliveira et al. (19).

Agastya et al. (20) used neutrophils isolated from three healthy subjects and exposed the neutrophils to non-acidified OME prodrug or acidified active OME, both at 0.5 mM. The treated neutrophils incubated with Saccharomyces cerevisiae for 10–60 min decreased total yeast phagocytosed with activated but not prodrug OME, but both prodrug OME and activated OME inhibited phagolysosome acidification. Haas et al. (21) used peripheral blood mononuclear cells (PBMCs) and granulocytes from 30 healthy volunteers (12 receiving PPIs and 18 not receiving PPIs) to assess the impact of PAN on PMN function. PMNs and whole blood samples were incubated with PAN 100 µM for 30 min. PAN inhibited interleukin (IL)-2, IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) and decreased phagocytosis, oxidative burst, and fMLP-induced migration. PMNs treated with prodrug PAN did not inhibit phagolysosomal acidification as demonstrated by increased levels of red fluorescence of DsRed E. coli or pHrodo Red E. coli particles when compared to bafilomycin A-treated cells. Bosnjak et al. (22) used RAW264.7 MΦs treated with LAN (range 1–10,000 nM/L) for 48 h to show concentration- and pH-dependent inhibition of the lysosomal cysteine proteases legumain and cathepsin B. LAN had no direct effect on phagolysosome acidification. Taken together, these studies demonstrate PPI inhibition of phagocytosis and phagosomal degradation. The acidification of lysosomes and phagolysosomal fusion are both dependent on V-ATPase function (23). These V-ATPases have been shown to be inhibited by disulfide bond formation (24), the same bond type that PPIs form to inhibit H⁺/K⁺ ATPases on parietal cells (25). Although Bosnjak et al. (22) did not find that treatment with LAN inhibited the acidification of lysosomal compartments, they did report that LAN directly inhibits the cysteine proteases cathepsin B and legumain through a direct interaction. This interaction was abolished through the addition of dithiothreitol and glutathione, reducers of disulfide bonds, suggesting that the inhibitory mechanism is the formation of a disulfide bond between the sulfhydryl group of LAN and the catalytic cysteine of the proteases.

Ritter et al. (26) used PMNs from healthy subjects to assess 30-min exposure to OME 10 μM on cell volume, intracellular pH, chemotaxis, and bacterial clearance. OME inhibited random and fMLP-induced migration and fMLP-induced cell swelling but did not inhibit bacterial clearance. Ohara and Arakawa (27) used PBMCs from 10 healthy subjects following a 2-day treatment with LAN 30 mg/day collected 7, 14, and 21 days after the start of treatment to show LAN-dependent decreased monocyte count and the number of cells expressing intracellular adhesion molecule (ICAM)-1. These alterations were not observed in cells treated with the H-2 inhibitor ranitidine, indicating a specificity for the PPI, LAN, in this mechanism. Yoshida et al. (28) used neutrophils from healthy adult men and treated the cells for 20 min with OME or LAN 10⁻⁶ to 10⁻⁴ mol/L followed by exposure to Helicobacter pylori water extract (HPE) or IL-1β. LAN and OME inhibited adhesion between neutrophils and epithelium following stimulation with HPE or IL-1β and decreased the expression of CD11b/CD18 and ICAM-1 in neutrophils. Haas et al. (21) used neutrophils isolated from healthy adult men. Cells were pretreated with OME or LAN 10⁻⁴ to 10⁻⁷ mol/L for 4 h prior to IL-8 or fMLP stimulation. LAN and OME were shown to inhibit the trans-endothelial migration of PMNs following stimulation with IL-8. LAN, but not OME, was shown to inhibit the increase in intracellular calcium that followed stimulation with fMLP. Martins De Oliveira et al. (19) used neutrophils collected from healthy donors and then exposed the cells to OME or PAN 10⁻⁶ to 10⁻⁴ mol/L to measure chemotaxis, cytotoxicity, and calcium mobilization. OME and PAN had no toxic effects on neutrophils. When IL-8 was added to PPI-treated cells, migration was inhibited which was reversed using nigericin, a K⁺ ionophore. PPI pretreatment reduced cytoplasmic calcium and p38 mitogen-activated protein kinase (MAPK) phosphorylation after stimulation of fMLP. Taken together, treatment with PPIs inhibits chemotactic migration and leukocyte recruitment likely via alteration of signaling and gene expression.

Koshio et al. (29) isolated PMNs from healthy adult donors and exposed the cells to LAN 1–5 µg/mL for 1 h. Cells were stimulated with IL-8, fMLP, or phorbol myristate acetate (PMA) to assess p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. LAN increased p38 MAPK phosphorylation (phospho-38) in a dose-dependent manner, but the effect was reduced in concentrations above 25 µg/mL. LAN treatment did not directly inhibit ERK1/2 and p38 MAPK phosphorylation, or ERK1/2 induced by IL-8, but inhibited the phosphorylation of ERK2 induced by PMA and the phosphorylation of p38 MAPK induced by fMLP. Balza et al. (30) used monocytes collected from four healthy donors and incubated the cells with OME 300 µM with LPS, zymosan, or both to assess the impact of OME on the secretion of IL-1β and TNF-α induced by pathogen-associated molecular patterns (PAMPs; LPS or zymosan). OME inhibited the secretion of IL-1β and TNF-α by 80% following LPS stimulation (IC₅₀: 100–300 µM). Monocytes were also collected from patients with cryopyrin-associated periodic syndrome (CAPS), an inflammatory autoimmune disease, to assess the impact of OME on IL-1β secretion. Treatment with OME also led to an 80% reduction in IL-1β secretion. The OME effect was not specific for TLR4 (receptor for LPS), as IL-8 was also OME-inhibited after stimulation by R848 (resiquimod) recognized by TLR7/8 and zymosan recognized by TLR2. OME did not directly inhibit IL-1β or TNF-α transcription or translation; rather, it inhibited K⁺ efflux, thus inhibiting the nucleotide-oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome assembly and caspase-1 activation preventing the generation of mature IL-1β.

The effects of esomeprazole (ESO) on inflammation were measured using a murine model of acute toxic shock sepsis. In the study of Balza et al. (30), mice were administered ESO 30 min before or after being given a lethal dose of LPS (12.5 mg/kg) and had a 40%–60% survival rate compared to 5% survival in the untreated group. LPS+ESO-treated mice were rechallenged with LPS of which 80% survived compared to 0% in the control mice. ESO-treated mice had significantly lower serum TNF-α, IL-1ß, and MΦs. The TLR agonism was also not specific for TLR4 as TNF-α and IL-1ß secretion was similarly reduced when stimulated with zymosan, a TLR2 agonist. The impact of ESO on non-infectious pathology was also assessed using sodium thioglycolate-induced peritonitis wherein mice injected with thioglycolates showed reduced infiltrating cells when compared to mice that received only thioglycolate. Thioglycolate+ESO mice also demonstrated reduced levels of macrophage inflammatory protein (MIP)-2 expressing neutrophils and monocyte chemoattractant protein (MCP)-1 expressing MΦs.

Sun et al. (31) treated human PBMCs in vitro with the prodrug OME 12.5–50 µg/mL for 4–24 h to assess cytokine production, phagolysosomal acidification, and transcriptomic analysis. OME inhibited TNF-α, MCP-1, IL-12, and IL-23 and the acidification of phagolysosomes. OME changed the transcriptional profile specifically of genes related to TLR signaling and cytokine signaling pathways. OME, LAN, PAN, and rabeprazole (RBP) inhibited the activation of nuclear factor (NF)-κB through suppression of the TLR4 signaling pathway. OME TLR4 inhibition was related to endosomal acidification specifically attenuating TLR signaling endosomes. This inhibition of TLR signaling was conserved across other endosomal signaling TLRs including TLR3 and TLR7/8 while having no effect on pathways activated by extracellular receptors TLR1/2. Taken together, these studies show PPI treatment altering signaling pathways, particularly TLR signaling, and altering p38 phosphorylation patterns.

Schulz-Geske et al. (32) used murine bone marrow-derived MΦs exposed to LAN 5–100 µM for 8 h to assess the expression of heme oxygenase (HO)-1 mRNA, ferritin mRNA, and ROS. LAN increased HO-1 mRNA, protein expression, ferritin, and ROS generation. Though HO-1 is induced by oxidative stress, preincubation with superoxide dismutase to alleviate oxidative stress did not reduce the increase in HO-1 expression. Ubagai et al. (33) isolated PMNs from healthy donors and then co-incubated the cells with LAN 0–10 µg/mL and LPS for 3 h. LAN had a negligible impact on cell viability. Chemokine receptor (CXCR)1/2, CD14, TLR4, TNF-α, and CD11b/CD18 mRNA transcripts were decreased in a dose- and time-dependent manner. Zhou et al. (34) treated wild-type C57/BL6 mice OME or ESO 10 mg/kg daily for 3 weeks prior to ischemic-reperfusion surgery. ESO but not OME caused a marked reduction in IL-1 and CD86 expression, but neither OME nor ESO changed the expression of CD206. Taken together, PPI displayed inhibitory effects on neutrophils and MΦs, especially M1 macrophages.