Inhibition of Human Neutrophil Functions by Sulfated and Nonsulfated Cholecystokinin Octapeptides

doi:10.1016/S0196-9781(96)00338-5

Peptides

Volume 18, Issue 3, 1997, Pages 415-422

https://doi.org/10.1016/S0196-9781(96)00338-5 Get rights and content

Abstract

Carrasco, M., M. Del Rio, A. Hernanz and M. De La Fuente. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides 18(3) 415–422, 1997.—The effects of CCK-8s and desulfated CCK-8 at concentrations ranging from 10⁻¹⁴ to 10⁻⁶M were studied in vitro on several functions of human peripheral neutrophils: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), ingestion of inert particles (latex beads) or cells (Candida albicans), and production of superoxide anion measured by the nitroblue tetrazolium reduction test. The effect of CCK-8s on intracellular levels of cAMP was investigated as well as the implication of calcium in the action of CCK-8s on phagocytic function using stimulants and inhibitors of both intracellular and extracellular calcium channels. The two peptides, at concentrations from 10⁻¹² to 10⁻⁸M, inhibited significantly both mobility and ingestion capacities and increased adherence to substrate. A dose-response relationship was observed with a maximum inhibition of neutrophil functions at 10⁻¹⁰M. CCK-8s and desulfated CCK-8 induced in these cells a significant, but transient, increase of cAMP levels at 60 s. Moreover, CCK-8s was found to inhibit completely the stimulation of latex bead phagocytosis in neutrophils produced by the calcium ionophore A23187. These results suggest that CCK-8 is a negative modulator of several neutrophil functions and that the inhibition of these activities could be carried out through an increase of the intracellular cAMP levels and a decrease of the extracellular calcium input.

Section snippets

Separation of Human Neutrophils

Peripheral blood was obtained from healthy men volunteers aged 22–34 years old. Blood was collected into vacuum EDTA-K3 tubes (Vacutainer, Becton-Dickinson). The time elapsed between collection of blood samples and cell isolation never exceeded 6 h. Peripheral blood polymorphonuclear (PMN) cells were separated by gradient sedimentation (density = 1.114) over Ficoll-Hypaque (Monopoly-Resolving-Medium, Flow) following a method previously described [36], and washed twice with phosphate-buffered

Results

The adherence index obtained for human peripheral blood neutrophils incubated with CCK-8s or desulfated CCK-8 is shown in Table 1. The two peptides increased significantly the adherence capacity of neutrophils at concentrations between 10⁻¹² and 10⁻⁸M, 10⁻¹¹ and 10⁻¹⁰M concentrations being the more efficient. Similar results (82 ± 7) were found for the calcium ionophore A23187 (10⁻⁷M) as a positive adherence control, whereas GRP (10⁻¹⁰M), used as a peptide control previously found by us to

Discussion

To our knowledge, the present study is the first carried out in relation to the in vitro effect of CCK peptides on human neutrophil functions. Peripheral blood neutrophils were used because these human immune cells are easily available in sufficient number and they are representative of the phagocytic population.

The effect of the CCK octapeptides was found, in general, at concentrations between 10⁻¹⁰ and 10⁻⁸M. These concentrations have been found in some tissues [1], whereas lower

Acknowledgements

We wish to thank M. Campos and J. Drummond, as well as Dr. Miquel, for their help during the realization of this study. We also thank Merck, Sharp & Dome for their gift of the CCK antagonist L364718. This work was supported by grant No. 92/0697 from Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss).

References (54)

J.E. Blalock
The syntax of immune-neuroendocrine communication
Immunol. Today
(1994)
M. De la Fuente et al.
Inhibition of murine macrophage functions by sulfated cholecystokinin octapeptide
Regul. Pept.
(1995)
M. De la Fuente et al.
Vasoactive intestinal peptide modulation of adherence and mobility in rat peritoneal lymphocytes and macrophages
Peptides
(1994)
M. De la Fuente et al.
Stimulation by vasoactive intestinal peptide (VIP) of phagocytic function in rat macrophages. Protein kinase C involvement
Regul. Pept.
(1993)
M. De la Fuente et al.
Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N
J. Neuroimmunol.
(1993)
M. Del Rio et al.
Chemoattractant capacity of bombesin, gastrin-releasing peptide and neuromedin C is mediated through PKC activation in murine peritoneal leukocytes
Regul. Pept.
(1994)
Y. Elitsur et al.
The inhibition effect of cholecystokinin in human colonic lamina propia lymphocyte proliferation, and reversal by the cholecystokinin receptor antagonist L-364817
Neuropeptides
(1991)
A.S. Fairhurst
Effect of ryanodine on skeletal muscle reticulum calcium adenosine triphosphatase (Ca-ATPase)
Biochem. Pharmacol.
(1973)
N. Hara et al.
Activation of single Ca²⁺-dependent K⁺ channel by external ATP in mouse macrophages
FEBS Lett.
(1990)
M.F. Lignon et al.
Cholecystokinin increases intracellular Ca²⁺ concentration in the human JURKAT T lymphocytes cell line
Eur. J. Pharmacol.
(1993)

M. Martínez et al.

Alterations in plasma and cerebrospinal fluid levels of neuropeptides in idiopathic senile anorexia

Regul. Pept.

(1993)

M.A. McMillen et al.

Cholecystokinin effect on human peripheral lymphocyte ionized calcium and mitogenesis

J. Surg. Res.

(1995)

J. Rehfeld

Immunochemical studies on cholecystokinin

J. Biol. Chem.

(1978)

A.B. Rodriguez et al.

Effects of cefmetazol, cefositin and imipenem on polymorphonuclear leukocytes

Gen. Pharmacol.

(1987)

M.R. Ruff et al.

Substance P receptor-mediated chemotaxis of human monocytes

Peptides

(1985)

P. Sacerdote et al.

Cholecystokinin and the immune system: Receptor-mediated chemotaxis of human and rat monocytes

Peptides

(1988)

P. Sacerdote et al.

Age-related changes of beta-endorphin and cholecystokinin in human and rat mononuclear cells

Peptides

(1991)

J.L. Scemama et al.

CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxin-sensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4-2J

FEBS Lett.

(1988)

C.P. Sung et al.

Modulation of U937 cell adhesion to vascular endothelial cells by cyclic AMP

Life Sci.

(1991)

S.C. Tang et al.

Regulation of human T lymphoblast growth by sensory neuropeptides: Augmentation of cholecystokinin-induced inhibition of Molt-4 proliferation by somatostatin and vasoactive intestinal peptide in vitro

Immunol. Lett.

(1992)

S.S. Yu et al.

β-Adrenergic receptor sequestration

J. Biol. Chem.

(1993)

T.E. Adrian et al.

Cholecystokinin

O. Bagasra et al.

Macrophage function in chronic experimental alcoholism. Modulation of surface receptors and phagocytosis

Immunology

(1988)

C.A. Baile et al.

Role of cholecystokinin and opioid peptides in control of food intake

Physiol. Rev.

(1986)

H. Besedovsky et al.

Network of immune-neuroendocrine interactions

Clin. Exp. Immunol.

(1977)

I. Bezprozvanny et al.

Bell-shaped calcium-response curves of Ins(1,4,5)P3 and calcium gated channels from endoplasmic reticulum of cerebellum

Nature

(1991)

S.V. Boyden

The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes

J. Exp. Med.

(1962)

Cited by (31)

Enteroendocrine cells-sensory sentinels of the intestinal environment and orchestrators of mucosal immunity
2018, Mucosal Immunology
The intestinal epithelium must balance efficient absorption of nutrients with partitioning commensals and pathogens from the bodies' largest immune system. If this crucial barrier fails, inappropriate immune responses can result in inflammatory bowel disease or chronic infection. Enteroendocrine cells represent 1% of this epithelium and have classically been studied for their detection of nutrients and release of peptide hormones to mediate digestion. Intriguingly, enteroendocrine cells are the key sensors of microbial metabolites, can release cytokines in response to pathogen associated molecules and peptide hormone receptors are expressed on numerous intestinal immune cells; thus enteroendocrine cells are uniquely equipped to be crucial and novel orchestrators of intestinal inflammation. In this review, we introduce enteroendocrine chemosensory roles, summarize studies correlating enteroendocrine perturbations with intestinal inflammation and describe the mechanistic interactions by which enteroendocrine and mucosal immune cells interact during disease; highlighting this immunoendocrine axis as a key aspect of innate immunity.
The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms
2008, Peptides
Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH₂) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH₂) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH₂, and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH₂, and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.
Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils
2008, Cell Calcium
The Ca²⁺-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca²⁺ acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H₂O₂, which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC₅₀) of 1 μM. Potentiation by Ca²⁺ is dose-dependent with an EC₅₀ of 300 nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100 nM), which significantly shifts the EC₅₀ for cADPR from 44 to 3 μM and for NAADP from 95 to 1 μM. TRPM2 activation by ADPR can be suppressed by AMP with an IC₅₀ of 10 μM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100 μM H₂O₂ enables subthreshold concentrations of ADPR (100 nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.
The first nonsulfated sulfakinin activity reported suggests nsDSK acts in gut biology
2007, Peptides
Invertebrate sulfakinins are structurally and functionally homologous to vertebrate cholecystokinin (CCK) and gastrin. To date, sulfakinins are reported to require a sulfated tyrosine for activity; sulfated and nonsulfated CCK and gastrin are active. This is the first nonsulfated sulfakinin activity reported. Nonsulfated Drosophila melanogaster sulfakinins or drosulfakinins (nsDSK I; PheAspAspTyrGlyHisMetArgPheNH2) and (nsDSK II; GlyGlyAspAspGlnPheAspAspTyrGlyHisMetArgPheNH2) decreased the frequency of contractions of adult D. melanogaster foregut (crop) in vivo. The EC50's for nsDSK I and nsDSK II were ∼2 × 10⁻⁹ M and ∼3 × 10⁻⁸ M, respectively. Nonsulfated DSK peptides also decreased the frequency of larval anterior midgut contractions. Sulfated DSK peptides decreased both adult and larval gut contractions. Whether sulfation is required for sulfakinin activity may depend on where the peptide is applied, what tissue is analyzed, or what preparation is used. D. melanogaster contains two sulfakinin receptors, DSK-R1 and DSK-R2; vertebrates contain two CCK receptors, CCK-1 and CCK-2. A sulfated DSK I analog, [Leu7] sDSK I, binds to expressed DSK-R1; the corresponding nonsulfated analog does not bind to DSK-R1. No DSK-R2 binding data are reported. Sulfated and nonsulfated CCK peptides preferentially bind to CCK-1 or CCK-2, respectively. Sulfated and nonsulfated sulfakinins may bind to DSK-R1 or DSK-R2, respectively. Sulfakinin activities, spatial and temporal distribution, and homology to CCK and gastrin suggest sulfated and nonsulfated DSK peptides act in diverse roles in the neural and gastrointestinal systems including gut emptying and satiety.
Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY
1998, Mechanisms of Ageing and Development
The general immunodepression found in ageing organisms may be related to changes in the neuroimmune network. In the present study, the migration capacity of lymphocytes from BALB/c mice of three different ages: young (12±2 weeks), adult (24±2 weeks) and old (72±2 weeks), has been assayed in vitro in response to three neuropeptides: sulfated cholecystokinin octapeptide (CCK-8s), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY) in a physiological range of concentrations (10⁻⁸–10⁻¹² M). The capacity of migration to a chemical gradient or chemotaxis was studied by the Boyden's technique using f-met-leu-phe at 10⁻⁸ M as chemoattractant. The results show a different response of lymphocytes to the different neuropeptides, as wells as to age, concentrations and locations studied. However, some similarities were found, for instance the three neuropeptides inhibited chemotaxis in thymus. The stimulatory effects that GRP and NPY exerted in young and adult mice were not observed in old animals. CCK-8s inhibited the chemotaxis in every organ studied, with the effect being more striking in old mice. Our conclusion is that stimulatory effects of the neuropeptides disappear or become inhibitory with ageing.
Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide
1998, Neuropeptides
The effects in vitro of the sulfated octapeptide form of cholecystokinin (CCK-8) at concentrations ranging from 10⁻¹³ M to 10⁻⁶M on several functions of murine lymphocytes were studied, i.e. adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis) and spontaneous and phytohemagglutinin (PHA)mediated proliferation. Lymphocytes were obtained from peritoneal suspension as well as from axillary nodes, spleen and thymus of BALB/c mice. CCK-8, at concentrations from 10⁻¹⁰ M to 10⁻⁸ M, significantly inhibited the mobility capacity and the PHA-induced proliferation and increased the adherence and the spontaneous proliferation of lymphocytes. A dose-response relationship was observed, with a maximum effect on lymphocyte functions at 10⁻¹⁰ M. In addition, CCK-8 induced a significant decrease in membrane and cytosol protein kinase C (PKC) activity in murine lymphocytes, as well as an increase of intracellular cyclic AMP levels. These results suggest that CCK-8 is a negative modulator of two important lymphocyte functions in the immune response, i.e. mobility and mitogen-induced proliferation, and that the PKC activity inhibition and cAMP increase could be the mechanisms through which CCK inhibits these lymphocyte activities.

View all citing articles on Scopus

¹: Requests for reprints should be addressed to Prof. M. De la Fuente.

View full text

Inhibition of Human Neutrophil Functions by Sulfated and Nonsulfated Cholecystokinin Octapeptides

Abstract

Section snippets

Separation of Human Neutrophils

Results

Discussion

Acknowledgements

Immunol. Today

Regul. Pept.

Peptides

Regul. Pept.

J. Neuroimmunol.

Regul. Pept.

Neuropeptides

Biochem. Pharmacol.

FEBS Lett.

Eur. J. Pharmacol.

Regul. Pept.

J. Surg. Res.

J. Biol. Chem.

Gen. Pharmacol.

Peptides

Peptides

Peptides

FEBS Lett.

Life Sci.

Immunol. Lett.

J. Biol. Chem.

Cholecystokinin

Macrophage function in chronic experimental alcoholism. Modulation of surface receptors and phagocytosis

Immunology

Role of cholecystokinin and opioid peptides in control of food intake

Physiol. Rev.

Network of immune-neuroendocrine interactions

Clin. Exp. Immunol.

Bell-shaped calcium-response curves of Ins(1,4,5)P3 and calcium gated channels from endoplasmic reticulum of cerebellum

Nature

The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes

J. Exp. Med.