Insulin-like growth factors enhance phagocytosis by human neutrophils in vitro

Regulatory Peptides, 49 (1993) 125-131 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00

125

REGPEP 01587

Insulin-like growth factors enhance phagocytosis by human neutrophils in vitro G u i - F a n g Jin a , , , Yan-Shi G u o

b,

Chuck Ball a and Clifford W. H o u s t o n a

aDepartment of Microbiology, University of Texas Medical Branch, Galveston, TX 77555-1019 (USA) and b Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555-1019 (USA) (Received 4 March 1993; revised version received 30 July 1993; accepted 9 August 1993)

Key words: Phagocytosis; Chemiluminescence; Insulin-like growth factor; EGTA; Nifedipine; G-protein

Summary

Polymorphonuclear neutrophils (PMNs) were isolated from human blood, and PMN phagocytosis was assessed by measuring the chemiluminescence (CL) response in the presence of ZAP (opsonized zymosin particles containing luminol). The administration of 6.5 nM of insulin-like growth factor I (IGF-I), des(1-3)-IGF-I, IGF-II or insulin to PMNs for 20 min resulted in significant increases of the CL response for all test preparations. Des(1-3)-IGF-I, a truncated IGF-I with low affinity binding to IGF binding proteins (IGFBPs), was the most potent CL stimulator. The CL production evoked by 6.5 nM of des(1-3)-IGF-I was inhibited significantly by both 0.25 and 1.0 mM of EGTA (Ca 2 ÷ chelator), or 10 #M nifedipine (Ca 2 + channel inhibitor), pertussis toxin (0.05 and 1.0 gg/ml) or cholera toxin (5/~g/ml). These results suggest that IGF-I and its homologues are potent stimulators of phagocytosis and that this action is modulated by IGFBP, and may require extracellular Ca 2 + and/or IGF-I receptor G-protein coupling.

Introduction

Insulin-like growth factors (IGFs) are a family of peptides that may influence growth, development and differentiation of a variety of cell types [ 1,2]. IGF-I (70 residues) and IGF-II (67 residues) are single-

*Corresponding author.

chain peptides of about 7.5 kDa with approx. 70~o sequence homology and 50~o homology with proinsulin. Unlike insulin, IGFs are found in the bloodstream associated with one of a group of specific IGF-binding proteins (IGFBPs) that are synthesized mainly in the liver [2,3]. The functions of IGFBPs may prolong the half-life of IGFs and modulate the role of IGFs in either inhibitory or stimulatory manners [2,3]. Recently, a truncated form of IGF-I,

126 des(1-3)-IGF-I, which lacked the aminoterminal tripeptide Gly-Pro-Glu, has been isolated from fetal and adult human brains as well as from bovine colostrum and porcine uterus [2,4]. Des(1-3)-IGF-I binds poorly to IGFBPs but can activate effectively the IGF-I receptors [2,4]. It has been reported that the action of des(1-3)-IGF-I is more potent in vitro and in vivo than IGF-I [2,4]. Although the growth-promoting effect of IGF-I has been shown in atrophied rat thymus [5], activated human lymphocytes [6,7], and human myeloid progenitors [8], the role(s) of IGFs in phagocytes is unclear at the present time. Several lines of evidence have shown that IGF-I receptors are present on human monocytes [9-13] and neutrophils [13] suggesting that IGF-I may affect the function of human phagocytes. In an attempt to define the effect of various molecular forms of IGFs on the phagocytic function of human polymorphonuclear leucocytes (PMNs) and the mechanism involved, we have examined the effect of IGFs on the chemiluminescence (CL) of human PMNs in vitro under various conditions. The measurement of CL has been demonstrated to be a useful method for the in vitro assessment of the function of phagocytosis [14] because actively phagocytizing phagocytes emit light (CL effect), an event that has been shown to be linked to the oxidative activity of phagocytes. Materials and Methods

PMN isolation Human PMNs were isolated using a neutrophil isolation kit (Los Alamos Diagnostics, Los Alamos, NM) from fresh blood collected from healthy adult volunteers. A 6 ml volume of heparinized human blood was layered onto 4 ml of neutrophil isolation medium and centrifuged at 400 g for 30 rain at room temperature. The PMN-rich fraction was collected, and a 2 ml of lysing buffer was added to destroy the contaminating RBCs. The cells then were washed twice with Hank's balanced salt solution (HBSS;

Gibco, Grand Island, NY) by centrifugation at 250 g for 20 rain at 20°C. The cells then were resuspended to a final concentration of 106 cells/ml in HB S S. The cells consisted of about 95 !~{,PM N identified by standard Wright stain. Cell viability was confirmed by trypan blue exclusion and always was greater than 95 ° o . Measurement of CL CL was measured as described previously [1416]. Aliquots of 10/~1 (106 cells/ml) of PMNs were incubated at 37°C for 20 min or as indicated, with 10 #1 of the following agents (final concentration): (1) 6.5 nM of IGF-I, IGF-II (Amgen, Thousand Oaks, CA), des( 1-3)-IGF-I (kindly provided by Genentech Inc., CA) and insulin (Sigma, St. Louis, MO); (2) various doses of des(1-3)-IGF-I, (ranging from 1.69.8 nM); (3) des(1-3)-IGF-I alone and in combination with EGTA (0.25 and 1.0 raM, Ca 2 + chelator) or nifedipine (10/~M, Ca 2+ channel inhibitor); (4) des(1-3)-IGF-I in the absence or presence of pertussis toxin (0.05 and 1.0 ~g/ml) or cholera toxin (CT, 5 gg/ml). At the end of incubation, CL of each sample was measured using a phagocytosis screening kit (Los Alamos Diagnostics, Inc., Los Alamos, NM). After addition of 200 #1 of ZAP (opsonized zymosan particles containing luminol), each tube was tapped gently and placed into a 20 ml liquid scintillation vial (23 x 58 ram). CL was measured immediately every 4.0 min for a total period of 40 min by a liquid scintillation analyzer (Packard Tri-Carb, Model 1500) using the 7th program which measures the light at room temperature. Data analysis CL values were standardized to the percentage of the peak of the control tube (designated as 100~';,). The integrated CL response (from 0 to 40 min) was calculated from the area beneath the curve from 4 - 6 observations and expressed as percent of control. Comparison of responses was evaluated statistically using the ANOVA test. A difference in P value of < 0.05 was considered statistically significant.

127

Results

Effect of incubation time on IGF-I-induced CL response The addition o f Z A P (oponized zymosan particles) to PMNs caused a CL response with a peak activity at 12 min in the control group (Fig. 1A); however, ZAP incubated with HBSS without PMNs did not produce a CL response (not shown). Treatment of PMNs with 6.5 nm des(1-3)-IGF-I resulted in a further increase of the CL response in comparison with the control group (Fig. 1A). The increase in the CL response was dependent on the length of incubation time of des(1-3)-IGF-I with PMNs. When PMNs were incubated with 6.5 nM of des(1-3)IGF-I (final concentration), for 0, 5, 10, 20 or 40 min, the optimal CL response was observed at 20 min incubation time (Fig. 1A). The integrated value of this CL response was 279 + 28~o of control (0-40 min) (Fig. 1B). Similar results for an optimal incubation time were obtained also from PMNs incubated with 6.5 nm of intact IGF-I (data not shown). Effect of IGF-I and its homologues on the CL response After incubation of PMNs with 6.5 nM IGF-I, des(1-3)-IGF-I, IGF-II and insulin for 20 min, the -~o 300 o

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40 min integrated CL responses to these peptides significantly increased when compared with the control (Fig. 2). Potency was established in decreasing order as follows: d e s ( 1 - 3 ) - I G F - I > I G F - I > I G F II > insulin. Moreover, the CL response to des(1-3)IGF-I (313 + 23 To of control) was much higher than that observed with the other homologues (Fig. 2). Effect of different concentrations of des(l-3)-IGF-I on CL Administration of des(1-3)-IGF-I at concentrations of 1.63, 3.27, 6.54 and 9.8 nM to PMNs for 20 min resulted in an increase of the CL response when compared with the control group and the 40 min integrated responses were 122 + 6, 152 + 3, 314 + 24 and 260 + 5 ~o of control, respectively. Three of these treated responses (3.27, 6.54 and 9.8 nM) were significantly higher than that observed in the control group (100 + 2~o, n = 5-6, P < 0 . 0 5 ) (Fig. 3). In addition, the integrated CL responses to 6.54 nM and 9.8 nM of des(1-3)-IGF-I also were significantly higher than that observed with 1.63 nM of des(13)-IGF-I. The 6.54 nM concentration of des(1-3)IGF-I appeared to result in the maximum CL response, since CL production at 9.8 nM of des(1-3)IGF-I declined when compared with the 6.54 nM group (Fig. 3).

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Minutes Fig. 1. (A) The time course of the chemiluminescence (eL) response after PMNs were incubated with des(1-3)-IGF-I at a concentration of 6.5 nM for 0 (control), 5, 10, 20 or 40 min. (B) The integrated CL responses to des(1-3)-IGF-I, which were calculated from the areas beneath the curves are shown in panel A. Each column is the mean _+S.E.M. from 5-6 observations and is expressed as percent of control. *P< 0.05 vs. control.

0

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Fig. 2. Comparison of the integrated CL response to 6.5 nM of IGF-I, des(l-3)-IGF-I (DES), IGF-II and insulin (INS) on phagocytosis. Each column is the mean _+S.E.M. from 4-6 independent observations and expressed as percent of control. *P< 0.05 vs. control.

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Fig. 3. Effect of various doses of des (1-3) IGF-I on CL responses in human PMNs in vitro. Each column represents the integrated CL response and is the mean + S.E.M. from 5-6 independent observations. P < 0.05 vs. control.

Effect of EGTA and nifedipine on des(1-3)-IGF-I induced CL We undertook the following study and used des(1-3)-IGF-I as a representative stimulator in order to determine a potential role for Ca 2 + in the CL response to IGF-I. The administration of 6.5 nM des(1-3)-IGF-I with 0.25 or 1.0 mM EGTA, resulted in a dramatic inhibition of des(1-3)-IGF-Istimulated CL activity (50 + 7~, and 43 + 3.5°~; of control, respectively) (Fig. 4). In order to assess the effect of a Ca 2 + -channel blocker on a des(1-3)-IGFI-elicited CL response, 10 # M nifedipine was added, which induced a 73~,o inhibition of the des(1-3)I G F - I stimulated CL response by P M N s (Fig. 4).

Fig. 4. Effect of EGTA and nifedipine (Nit) on the CL response stimulated by des(l-3)-IGF-I (DES, 6.5 nM). Each column is the integrated CL response over a 0-40 min period and is the mean _+S.E.M. from 4-5 independent observations. *P < 0.05 vs des(1-3)-lGF-I alone.

were significantly lower than that in control (68.7 + 5~o and 53.2 + 1.8% of control after pretreatment with 0.05 and 1.0 #g/ml of PT, respectively) (Fig. 5). Similarly, P M N s pretreatment with CT at 5 #g/ml for 2 h resulted in a significant inhibition of the CL response to des(1-3)-IGF-I (30 + 2% of control) (Fig. 5).

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Effects of PT and CT on des(1-3)-IGF-I stimulated CL We have investigated the effects of PT (pertussis toxin) or CT (cholera toxin) treatment on the CL response to des(1-3)-IGF-I to understand the possible involvement of guanine nucleotide-binding proteins (G-proteins) in the transmembrane signaling process for I G F - I action. P M N s were treated with 0.05 and 1.0 ~g/ml of PT for 2 h at 37°C. In a preliminary study, PT alone did not alter the CL activity of phagocytes (data not shown). After PT treatment, P M N s were incubated further with 6.5 m M d e s ( 1 - 3 ) - I G F - I for 20 min. The CL responses

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Fig. 5. Effect of pertussis toxin (PT) and cholera toxin (CT) on the CL response evoked by des(l-3)-IGF-I (DES, 6.5 nM). Each column is the integrated CL response over a 0-40 min period and is expressed as the mean+ S.E.M. from 4-6 observatons. *P<0.05 vs. des(1-3)-IGF-I alone.

129 Discussion

The present findings show that IGF-I and its homologues, including des(1-3)-IGF-I, IGF-II and insulin, significantly stimulated the CL response, an index that represents the phagocytic activity of PMNs [ 14]. These observations are similar to those of Fu et al. [ 17], who demonstrated that IGF-I and growth hormone primed human and porcine neutrophils in vitro to result in enhanced O f production, another method of evaluating the phagocytic capacity of neutrophils. The current study further demonstrated that a trucared IGF-I, des(1-3)-IGF-I, is the most potent stimulant among IGF homologues studied for enhancement the CL response by PMNs. In a number of reports, the action of des(1-3)-IGF-I has been shown to be more potent than IGF-I in vitro and in vivo in the presence of IGFBPs [2,4]; whereas, in the absence of endogenous or added IGFBPs, the two peptides have nearly equal activities [ 18]. This result suggests that IGFBP(s) may inhibit the CL activity of phagocytes in response to IGF-I. Since the experiments mentioned above were conducted under serum-flee conditions, the IGFBP(s) that depressed the IGF-I stimulated CL response by phagocytes did not originate from serum during the experiment. There are two possibilities to explain the original source of IGFBP(s): (1) the I G F B P may associate on the cell membranes of PMNs before isolation of the cells; (2) the IGFBP(s) may be secreted by phagocytes. The latter possibility has been confirmed by Lee and coworkers [19] who demonstrated that IGFBP-2 mRNA is present in activated macrophages. Phagocytosis is a phenomenon of engulfment of a microbial agent by phagocytes [20]. This process is due to the polymerization of the actin skeleton of phagocytes and changes in cell shape. The actin polymerization is evoked by gelsolin, an actin-modulating protein, that is activated by elevated intracellular Ca 2 + [21]. The increased cytosolic concentration of free Ca 2 + may result from either the

Ca 2 + released from intracellular stores or the influx of extracellular Ca 2 + [21,22]. The role of extracellular Ca 2+ in IGF-I-stimulated CL response was examined by use of the divalent cation chelator, EGTA. Reportedly, E G T A does not penetrate cell membranes [23], can bind extracellular Ca 2+, can remove the Ca 2 + bound to the surface of cell membranes [23], and may deplete intracellular Ca 2+ by binding Ca 2 + that is effluxed into the interstitial space [24]. The finding that EGTA at concentrations of 0.25 and 1.0 mM significantly inhibited the des(13)-IGF-I stimulated CL response suggests that inhibition of Ca 2 + transport into the phagocytes resulted in an interruption of an essential mediator for the stimulus-response process. Nifedipine, a Ca 2 + channel inhibitor, at 10 #M also obviously inhibited the CL response to des(1-3)-IGF-I, indicating that this CL response requires entry of the extracellular Ca 2 + through the Ca 2 + channels. However, the question of whether des(1-3)-IGF-I also elicits mobilization of Ca 2+ from intracellular stores requires further study. Guanine nucleotide-binding proteins (G-proteins) are a class of membrane-associated proteins that couple the activation of hormone receptors to stimulation of effector enzymes and ion channels [2527]. The G-protein family include the stimulatory (Gs) and inhibitory (Gi) G-proteins of the hormonesensitive adenylate cyclase system, the transducin (Gt) of vertebrate retina that communicates between light activation of rhodopsin and stimulation of cGMP-dependent phosphodiesterase, and a fourth member (Go) that has been identified in brain tissue without known function [25-27]. G-proteins are composed of 2, fl, and ? subunits. The 2-subunit contains a single guanine nucleotide-binding site with high affinity and a site for NAD-dependent ADPribosylation catalyzed by cholera toxin (CT) or pertussis toxin (PT). It has been demonstrated that CT stimulates adenylate cyclase by ADP-ribosylating the ~-subunit of Gs, whereas PT inactivates the Gi by modifying the ~-subunit of Gi, both processes resulting in an increase of intracellular cAMP [25-27].

130

Reportedly, multiple functions of monocytes and PMNs are regulated by G proteins [28-32]. In order to know whether the stimulation of phagocytosis by des(1-3)-IGF-I is also mediated by G-protein, the effect of PT or CT on phagocytic function stimulated by des (1-3)-IGF-I was examined in this study. In contrast to those studies in which PT and CT have different roles on the regulation of adenylate cyclase [25,26], we found that PT and CT both significantly depressed the phagocytosis response elicited by treatment of PMNs with des(1-3)-IGF-I. However, these results are analogous with the report of Gresham etal. [33], who showed that a cytokinestimulated (YM-10E) and amphotericin B-stimulated ingestion is completely abrogated by treating the PMNs with either PT or CT and that the effect of PT-and CT on the PMN phagocytic function is independent of adenylate cyclase stimulation. Although Gi and Gs contain sites only for ADP-ribosylation by PT and CT, respectively, other G-proteins, such as transducin and Go, are capable of being ADPribosylated by both toxins [25-27]. In addition, a novel G-protein present in PMNs has been shown to possess both PT- and CT-ADP-ribosylation sites [34]. Therefore, the phagocytosis stimulated by des(1-3)-IGF-I, may not be regulated by Gs or Gi, but could be mediated by some other yet unidentified G-protein. Further study is required to define this presumed G-protein. Since IGFs exist in human blood [ 1] and may be synthesized and released from human macrophages [35,36], it is possible that phagocytosis by PMNs may be stimulated by IGFs via a autocrine, paracrine or endocrine mechanism.

Acknowledgements The authors would like to thank the Genentech, Inc., San Francisco, CA for gifts of des(1-3)-IGF-I. Dr. Howard L. Foyt's helpful suggestion served to improve this manuscript. Our gratitude is extended to Rosalind Lopez and Charlene Hoff for typing this manuscript.

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