Helen M. Korchak

Protein kinase C isotypes and signal-transduction in human neutrophils: Selective substrate specificity of calcium-dependent β-PKC and novel calcium-independent nPKC

Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.

Selective Role for β-Protein Kinase C in Signaling for O⨪2 Generation but Not Degranulation or Adherence in Differentiated HL60 Cells

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete β-PKC, both βI- and βII-PKC, but not α-PKC, δ-PKC, or ζ-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of β-PKC in dHL60 cells elicited selective inhibition of O⨪2generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited β-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by β-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in β-PKC-depleted dHL60 cells. Phosphorylation of p47 phox and translocation of p47 phox to the membrane are essential for activation of the NADPH oxidase and generation of O⨪2. β-PKC depletion had no effect on the level of p47 phox in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47 phox to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in β-PKC-depleted cells. These results indicate that β-PKC is essential for signaling for O⨪2 generation but not cell adherence or azurophil degranulation. Depletion of β-PKC inhibited ligand-induced phosphorylation of p47 phox , translocation of p47 phox to the membrane, and activation of O⨪2 generation.

Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of δ‐PKC and ERK1/2

TNF is implicated in the suppression of neutrophil apoptosis during sepsis. Multiple signaling pathways are involved in TNF‐mediated antiapoptotic signaling; a role for the MAP kinases (MAPK), ERK1/2, and p38 MAPK has been suggested. Antiapoptotic signaling is mediated principally through TNF receptor‐1 (TNFR‐1), and the PKC isotype‐delta (δ‐PKC) is a critical regulator of TNFR‐1 signaling. δ‐PKC associates with TNFR‐1 in response to TNF and is required for NFκB activation and inhibition of caspase 3. The role of δ‐PKC in TNF‐mediated activation of MAPK is not known. The purpose of this study was to determine whether the MAPK, ERK1/2, and p38 MAPK are involved in TNF antiapoptotic signaling and whether δ‐PKC is a key regulator of MAPK activation by TNF. In human neutrophils, TNF activated both p38 MAPK and ERK1/2 principally via TNFR‐1. The MEK1/2 inhibitors PD098059 and U0126, but not the p38 MAPK inhibitor SB203580, decreased TNF antiapoptotic signaling as measured by caspase 3 activity. A specific δ‐PKC antagonist, V1.1δ‐PKC‐Tat peptide, inhibited TNF‐mediated ERK1/2 activation, but not p38 MAPK. ERK1/2 inhibition did not alter recruitment of δ‐PKC to TNFR‐1, indicating δ‐PKC is acting upstream of ERK1/2. In HL‐60 cells differentiated to a neutrophilic phenotype, δ‐PKC depletion by δ‐PKC siRNA resulted in inhibition of TNF mediated ERK1/2 activation but not p38 MAPK. Thus, ERK1/2, but not p38 MAPK, is an essential component of TNF‐mediated antiapoptotic signaling. In human neutrophils, δ‐PKC is a positive regulator of ERK1/2 activation via TNFR‐1 but has no role in p38 MAPK activation.

Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10−6 M)-induced Ca2+ increase. In contrast, the addition of SP (10−6 M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Roles for βII-Protein Kinase C and RACK1 in Positive and Negative Signaling for Superoxide Anion Generation in Differentiated HL60 Cells

β-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O⨪2). Neutrophils and neutrophilic HL60 cells contain both βI and βII-PKC, isotypes that are derived by alternate splicing. βI-PKC-positive and βI-PKC null HL60 cells generated equivalent amounts of O⨪2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of βII-PKC from βI-PKC null cells inhibited ligand-initiated O⨪2generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47 phox , with βII-PKC but not with RACK1, a binding protein for βII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of β-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O⨪2 generation. Therefore, βII-PKC but not βI-PKC is essential for activation of O⨪2generation and plays a positive role in signaling for NADPH oxidase activation in association with p47 phox . In contrast, RACK1 is involved in negative signaling for O⨪2 generation. RACK1 binds to βII-PKC but not with the p47 phox ·βII-PKC complex. RACK1 may divert βII-PKC to other signaling pathways requiring β-PKC for signal transduction. Alternatively, RACK1 may sequester βII-PKC to down-regulate O⨪2 generation.

Impaired metabolic function and signaling defects in phagocytic cells in glycogen storage disease type 1b.

Patients with glycogen storage disease (GSD) type 1b (1b), in contrast to patients with GSD type 1a (1a), are susceptible to recurrent bacterial infections suggesting an impairment in their immune system. In this study, phagocytic cell (neutrophil and monocyte) respiratory burst activity, as measured by superoxide anion generation, oxygen consumption, and hexose monophosphate shunt activity, was markedly reduced in both neutrophils and monocytes from GSD 1b patients as compared with either GSD 1a patients or healthy adult control cells. Degranulation, unlike respiratory burst activity, was not significantly different in neutrophils from GSD 1b patients as compared with controls. Both neutrophils and monocytes from GSD 1b patients showed decreased ability to elevate cytosolic calcium in response to the chemotactic peptide f-Met-Leu-Phe. In addition, calcium mobilization in response to ionomycin was also attenuated suggesting decreased calcium stores. Thus, reduced phagocytic cell function in GSD 1b is associated with diminished calcium mobilization and defective calcium stores. Defective calcium signaling is associated with a selective defect in respiratory burst activity but not degranulation.

Regulation of TNF-induced oxygen radical production in human neutrophils: role of δ-PKC

In human neutrophils, TNF‐elicited O2− production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O2− generation has yet to be fully elucidated. Previously, we identified δ‐PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of δ‐PKC with a dominant‐negative δ‐PKC TAT peptide resulted in a significant delay in the onset time of TNF‐elicited O2− generation but had no effect on Vmax, indicating an involvement of δ‐PKC in the initiation of O2− production. In contrast, fMLP‐elicited O2− production in adherent and nonadherent neutrophils was δ‐PKC‐independent, suggesting differential regulation of O2− production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time‐dependent association of δ‐PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for δ‐PKC in regulating O2− production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF‐elicited O2− generation. TNF‐mediated ERK but not p38 MAPK recruitment to p47phox was δ‐PKC‐dependent. δ‐PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of δ‐PKC (Ser643) and δ‐PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K‐dependent process. Thus, signaling for TNF‐elicited O2− generation is regulated by δ‐PKC. Adherence‐dependent cooperative signaling activates PI3K signaling, δ‐PKC phosphorylation, and δ‐PKC recruitment to p47phox. δ‐PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.

Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R)

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP‐preferring receptor neurokinin‐1 receptor (NK1R) has two forms: a full‐length NK1R (NK1R‐F) isoform and a truncated NK1R (NK1R‐T) isoform, which lacks the terminal cytoplasmic 96‐aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R‐T but not NK1R‐F. SP interactions with NK1R‐T did not mobilize calcium (Ca2+), but SP mobilized Ca2+ when the NK1R‐F was transfected into monocytes. However, the NK1R‐T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5‐elicited Ca2+ mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R‐T also enhanced CCL5‐mediated chemotaxis, which was ERK1/2‐dependent. NK1R‐T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R‐T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R‐T is functional in human monocytes and activates select signaling pathways, and the NK1R‐T‐mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain.

In vitro and in vivo effects of granulocyte colony-stimulating factor on neutrophils in glycogen storage disease type 1B: granulocyte colony-stimulating factor therapy corrects the neutropenia and the defects in respiratory burst activity and Ca2+ mobilization.

ABSTRACT: Children with glycogen storage disease (GSD) type 1b are susceptible to recurrent bacterial infections and have chronic neutropenia accompanied by phagocytic cell dysfunction including decreased superoxide anion (O2−) generation, calcium (Ca2+) mobilization, and chemotactic activity. Granulocyte colony-stimulating factor (G-CSF), a cytokine that corrects neutropenia in other diseases, in vitro enhances f-Met-Leu-Phe-triggered neutrophil O2− generation. Short-term pretreatment (15 min) of GSD 1b neutrophils with G-CSF increased the rate of O2− production (p < 0.01); however, this rate was still significantly below the rate of O2− production in control neutrophils. Recombinant human G-CSF (5 μg/kg/d) was administered s.c. to a GSD 1b patient. Before treatment, absolute neutrophil counts were < 500/mm3. Two d after G-CSF administration, the absolute neutrophil counts increased to 1333 and remained in the normal range during a 12-mo follow-up period. In vivo, G-CSF therapy increased f-Met-Leu-Phe-stimulated O2− production to 52% of control after 1 mo, and by mo 4, O2− production reached control levels. Our previous studies (J Clin Invest 56:196–202, 1990) demonstrated that decreased O2− production in neutrophils was associated with impaired Ca2+ mobilization. In vivo administration of G-CSF increased f-Met-Leu-Phe-triggered Ca2+ mobilization by neutrophils to 43% of control by mo 1 of G-CSF therapy and to 93% of control by mo 4, thus paralleling the improvements in O2− generation. In contrast, G-CSF therapy had no effect on the defective neutrophil chemotaxis. In summary, G-CSF therapy produced a rapid increase in circulating neutrophils and a gradual correction of O2− production. Long-term exposure to G-CSF may be required for correction of both neutropenia and O2− production in GSD 1b patients.

Protection against sepsis-induced lung injury by selective inhibition of protein kinase C-δ (δ-PKC)

Inflammation and proinflammatory mediators are activators of δ‐PKC. In vitro, δ‐PKC regulates proinflammatory signaling in neutrophils and endothelial and epithelial cells, cells that can contribute to lung tissue damage associated with inflammation. In this study, a specific δ‐PKC TAT peptide inhibitor was used to test the hypothesis that inhibition of δ‐PKC would attenuate lung injury in an animal model of ARDS. Experimental ARDS was induced in rats via 2CLP, a model of polymicrobial sepsis. Following 2CLP surgery, the δ‐PKC TAT inhibitory peptide (2CLP+δ‐PKC TAT in PBS) or PBS (2CLP+PBS) was administered intratracheally. Controls consisted of SO, where animals underwent a laparotomy without 2CLP. Twenty‐four hours after SO or 2CLP, blood, BALF, and lung tissue were collected. 2CLP induced δ‐PKC phosphorylation in the lung within 24 h. Treatment with the δ‐PKC TAT inhibitory peptide significantly decreased pulmonary δ‐PKC phosphorylation, indicating effective inhibition of δ‐PKC activation. Plasma and BALF levels of the chemokines CINC‐1 and MIP‐2 were elevated in 2CLP + PBS rats as compared with SO rats. Treatment with δ‐PKC TAT reduced 2CLP‐induced elevations in chemokine levels in BALF and plasma, suggesting that δ‐PKC modulated chemokine expression. Most importantly, intratracheal administration of δ‐PKC TAT peptide significantly attenuated inflammatory cell infiltration, disruption of lung architecture, and pulmonary edema associated with 2CLP. Thus, δ‐PKC is an important regulator of proinflammatory events in the lung. Targeted inhibition of δ‐PKC exerted a lung‐protective effect 24 h after 2CLP.