Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils

doi:10.1016/0898-6568(92)90019-5

Cellular Signalling

Volume 4, Issue 5, September 1992, Pages 501-509

https://doi.org/10.1016/0898-6568(92)90019-5 Get rights and content

Abstract

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compare favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells)_to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca²⁺ concentration of 1 μM, a value of the cytosolic free Ca²⁺ concentration occurring in formymethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-γ-S besides 1 μM Ca²⁺ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.

References (30)

P. Bellavite
Free Rad. Biol. Med.
(1988)
H.M. Korchak et al.
J. biol. Chem.
(1984)
H.W.M. Niessen et al.
Cellular Signalling
(1991)
J.E. Smolen et al.
Biochim. biophys. Acta
(1990)
M.J. Metzelaar et al.
CD63 antigen
J. biol. Chem.
(1991)
A.J. Verhoeven et al.
Blood
(1988)
S. Grinstein et al.
J. biol. Chem.
(1988)
T.W. Kuijpers et al.
Blood
(1991)
C.R. Jost et al.
Blood
(1991)
G.P. Smith et al.
Biochim. biophys. Acta
(1982)

B. Dewald et al.

Biochim. biophys. Acta

(1986)

J.E. Merritt et al.

J. biol. Chem.

(1989)

J.E. Smolen et al.

Biochim. biophys. Acta

(1986)

M. Lindau et al.

Biochim. biophys. Acta

(1991)

H.L. Malech et al.

New Engl. J. Med.

(1987)

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To examine the phenotype of leukocytes from CF patients with the G551D mutation, blood was stained for flow cytometric analysis before ivacaftor treatment and at one and six months post drug treatment. Included in the staining cocktails were markers of priming/activation including CD63 [a marker of degranulation [16]], the activation-specific epitope of CD11b [no cross-reactivity with native CD11b molecules on non-activated PMNs and monocytes [17]], and intracellular activity of the inflammasome-associated enzyme caspase-1 [18], which leads to the production of the potent pro-inflammatory cytokine interleukin-1β. The baseline levels of these markers before patients began treatment with ivacaftor were higher than levels found in age, sex, and race-matched healthy controls, and these levels decreased over the course of ivacaftor treatment.
Ivacaftor improves clinical outcome by potentiation of mutant G551D CFTR. Due to the presence of CFTR in monocytes and polymorphonuclear neutrophils (PMNs), we hypothesized that ivacaftor may impact leukocyte activation.
We examined blood leukocytes from G551D CF subjects prior to and at one and six months after receiving ivacaftor. Blood leukocytes from ivacaftor-naïve G551D, F508del, and healthy controls were also treated with ivacaftor ex vivo to assess mutation-specific effects.
Compared to healthy controls, G551D CF subjects had significantly higher expression of active CD11b on PMNs and of CD63 on monocytes, which were normalized by in vivo ivacaftor treatment. Ex vivo exposure to ivacaftor of blood cells from G551D, but not F508del and healthy subjects, resulted in changes in CXCR2 and CD16 expression on PMNs.
In vivo and ex vivo exposure of G551D CF leukocytes to ivacaftor resulted in an altered activation profile, suggesting mutation-specific leukocyte modulation.
A neutrophil intrinsic impairment affecting Rab27a and degranulation in cystic fibrosis is corrected by CFTR potentiator therapy
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This defect was also observed after stimulation with fMLF or fMLF in combination with TNF-α, indicating that the observed degranulation impairment was not stimuli specific (supplemental Figure 4). As an alternative approach, we investigated the membrane expression of CD66b.44 As illustrated in Figure 2F, up-regulation of CD66b to the plasma membrane was greatly decreased in CF neutrophils compared with HC cells (n = 8; P = .004, P = .01, and P = .004 after 5, 10, and 20 minutes, respectively).
Studies have endeavored to reconcile whether dysfunction of neutrophils in people with cystic fibrosis (CF) is a result of the genetic defect or is secondary due to infection and inflammation. In this study, we illustrate that disrupted function of the CF transmembrane conductance regulator (CFTR), such as that which occurs in patients with ∆F508 and/or G551D mutations, correlates with impaired degranulation of antimicrobial proteins. We demonstrate that CF blood neutrophils release less secondary and tertiary granule components compared with control cells and that activation of the low-molecular-mass GTP-binding protein Rab27a, involved in the regulation of granule trafficking, is defective. The mechanism leading to impaired degranulation involves altered ion homeostasis caused by defective CFTR function with increased cytosolic levels of chloride and sodium, yet decreased magnesium measured in CF neutrophils. Decreased magnesium concentration in vivo and in vitro resulted in significantly decreased levels of GTP-bound Rab27a. Treatment of G551D patients with the ion channel potentiator ivacaftor resulted in normalized neutrophil cytosolic ion levels and activation of Rab27a, thereby leading to increased degranulation and bacterial killing. Our results confirm that intrinsic alterations of circulating neutrophils from patients with CF are corrected by ivacaftor, thus illustrating additional clinical benefits for CFTR modulator therapy.
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Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.
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Granulocytes are defined as the population of granulated white blood cells (eosinophils, neutrophils, and basophils). These cells are involved in inflammation and contribute to the pathogenesis of allergic and inflammatory diseases. Inflammation is induced by the release of mediators from granulocytes recruited to or resident within tissues, resulting in edema, leukocyte recruitment, and tissue injury. Eosinophils and neutrophils express Rac1 and Rac2 guanosine triphosphatases (GTPases), 2 members of the Rho GTPase subfamily of ras-related GTPases. Rho GTPases are activated by receptors in the cell membrane and are proposed to function as intracellular molecular switches to regulate mediator release, including exocytosis, from granulocytes. Exocytosis involves granule fusion, which requires the binding of intracellular membrane receptors known as SNAP receptor (SNAREs; soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors). Eosinophils and neutrophils express similar SNARE isoforms that are important in granule fusion events. Together, these molecules link together to form a common signaling pathway for mediator release from granulocytes. Identifying these molecules and their function may provide novel targets for the prevention of inflammatory reactions.
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Citation Excerpt :
This concurs with the inhibitory effect of roscovitine on the GTP-stimulated increase in surface expression of CD63 and CD66b in neutrophils. This increase in plasma membrane expression of CD63 and CD66b has been shown in the past to closely correlate with conventional assays of secretion from neutrophil azurophil and specific granules, respectively (30, 31). Although roscovitine may cause some inhibitory effect on the cell cycle Cdk1 and Cdk2, such effect may be disregarded in secretion by terminally differentiated neutrophils.
We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35^–/– mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.
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Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-β1 (TGF-β1) has been implicated in the development of renal fibrosis accompanied by α-smooth muscle actin (α-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-β1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-β1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-α1 or anti-α2 integrin subunit antibodies significantly suppressed TGF-β1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-β1 enhanced the formation of the collagen fibrils by cell attachment to collagen via α1β1 and α2β1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-β1 enhanced cell adhesion to collagen via the increased expression of α1 and α2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-β1. Furthermore, TGF-β1 increased the expression of a putative contractile protein, α-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-β1 stimulates fibroblast–collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and α-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.

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Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils

Abstract

Free Rad. Biol. Med.

J. biol. Chem.

Cellular Signalling

Biochim. biophys. Acta

J. biol. Chem.

Blood

J. biol. Chem.

Blood

Blood

Biochim. biophys. Acta

Biochim. biophys. Acta

J. biol. Chem.

Biochim. biophys. Acta

Biochim. biophys. Acta

New Engl. J. Med.