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Endocrinology Vol. 143, No. 4 1206-1212
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

IGF-I Inhibits Spontaneous Apoptosis in Human Granulocytes

Ron Kooijman, Astrid Coppens and Elisabeth Hooghe-Peters

Department of Pharmacology, Medical School, Vrije Universiteit Brussel, B-1090 Brussels, Belgium

Address all correspondence and requests for reprints to: Ron Kooijman, Department of Pharmacology, Medical School, Free University of Brussels (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: . rkooi{at}farc.vub.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Granulocytes are key cells in inflammatory processes that are recruited to sites of inflammation by chemoattractants such as IL-8 produced by neutrophils and monocytes. Programmed cell death (apoptosis) of granulocytes and subsequent recognition and phagocytosis by macrophages is a crucial mechanism for resolution of inflammation. Because IGF-I is a potent antiapoptotic factor, we addressed the effects of IGF-I on in vitro apoptosis of human peripheral blood granulocytes. We detected 1390 ± 467 IGF-I receptors with a dissociation constant of 2.3 ± 0.9 nM on purified granulocytes. Using microscopical analysis, annexin V binding assays to detect relocation of phosphatidylserine to the cell surface, and DNA fragmentation assays, we showed that IGF-I inhibits spontaneous apoptosis of granulocytes in serum-free culture by 32–45%. IGF-I did not modulate the secretion of IL-6, TNF{alpha}, and IL-8 by granulocytes, but IL-8 secretion by peripheral blood mononuclear cells was enhanced by 40%. These observations indicate that IGF-I may promote granulocyte functions by increasing granulocyte longevity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUTROPHILIC AND EOSINOPHILIC granulocytes are short-lived terminally differentiated blood cells that play a pivotal role in inflammatory responses. Neutrophils are phagocytic cells that participate in inflammatory reactions as a first line of defense against invading microorganisms. Eosinophils have important antiparasitic functions and are involved in allergic inflammation. Granulocytes are rapidly recruited to inflammatory sites by chemotactic factors. Neutrophils produce chemoattractant factors like IL-8, growth-related gene product-{alpha}, and macrophage inflammatory protein-1{alpha} and -1ß (1). IL-8 is one of the most abundant cytokines produced by neutrophils that also triggers degranulation and oxidative burst, both of which contribute to destructive properties of these cells (1). Cellular homeostasis and the resolution of granulocyte-mediated inflammatory reactions are crucial to prevent tissue damage. Granulocyte apoptosis and subsequent recognition and phagocytosis of these cells by macrophages is considered as a mechanism to control inflammatory reactions (2).

IGF-I plays a key role in embryonic and postnatal growth, exerts metabolic effects, and is involved in tissue homeostasis through regulation of cell proliferation and programmed cell death (apoptosis). Evidence suggests that IGF-I affects homeostasis in the immune system by enhancing lymphopoiesis, granulopoiesis, cell proliferation, and cell survival (3, 4). For instance, IGF-I inhibits apoptosis of T cells activated by phytohemagglutinin (5) or through specific stimulation of CD3 and CD28 (6). Furthermore, IGF-I inhibits apoptosis of the promyeloid leukemic cell line HL-60 and allows it to differentiate toward granulocytes (7). These antiapoptotic effects, and the stimulating effects of IGF-I on cytokine secretion by peripheral blood mononuclear cells (PBMCs) (8, 9, 10) may lead to modulation of inflammatory reactions. An intriguing question is whether IGF-I affects inflammatory responses through effects on granulocyte longevity and cytokine secretion by these cells.

Although IGF-I has been reported to enhance granulocyte functions such as phagocytosis, degranulation, and oxidative burst (11, 12), the effects of IGF-I on apoptosis of primary granulocytes and cytokine production by these cells have not been established. We report that IGF-I inhibits apoptosis of freshly isolated peripheral blood granulocytes as assessed by microscopical analysis, annexin V staining, and DNA fragmentation assays. The secretion of IL-8, IL-6, and TNF{alpha} by granulocytes is not affected by IGF-I, indicating that the inhibiting effects of IGF-I on granulocyte apoptosis are not mediated by autocrine cytokines. In contrast, IGF-I stimulates the secretion of IL-8, and to a lesser extend, the secretion of IL-6 and TNF{alpha} by PBMC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human (rh) IGF-I was kindly provided by Lilly Research Laboratories (Indianapolis, IN). Contamination with endotoxin was not detectable using the Limulus Amebocyte Lysate assay (detection limit 1.25 pg/µg; BioWhittaker, Inc., Walkersville, MD). 125INa was from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Granulocyte-monocyte colony stimulating factor (GM-CSF) was purchased from Novartis Pharma (Basel, Switzerland). Insulin, lipopolysaccharide (LPS) from Salmonella typhosa, chloramine-T, and BSA were purchased from Sigma (Bornem, Belgium), and {alpha}IR3 was obtained from Oncogene (Darmstadt, Germany). Lymphoprep was from Nycomed Pharma (Oslo, Norway), and Roswell Park Memorial Institute medium, penicillin and streptomycin were obtained from Life Technologies, Inc. (Merelbeke, Belgium).

Subjects
Blood donors were between 25 and 55 yr of age. Informed consent was obtained from all blood donors and the research protocol has been approved by the local ethical committee. The number of female and male subjects for each experiment are indicated in the legends. Different subjects were used for IGF-I binding studies, apoptosis assays, and cytokine assays. Morphological analysis and annexin V binding studies to assess apoptosis were done in one single experiment using one group of five donors. Three of these donors took part in an assay with six subjects to measure apoptosis using DNA fragmentation assays.

Cell preparation and cell culture
Human granulocytes and PBMC were purified from heparinized venous blood drawn from healthy donors. Granulocytes were separated from PBMC by centrifugation on Lymphoprep as described before (13). The contamination in these preparations with other cells was less than 2%, and cell viability as assessed by trypan blue exclusion was always higher than 95%.

Freshly isolated cells were suspended at a density of 106 cells/ml in serum-free medium (Roswell Park Memorial Institute 1640 with glutamax-I, supplemented with 0.02% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin), and cultured in 5 ml polystyrene Falcon tubes (Becton Dickinson and Co., Erembodegem, Belgium) in a humidified 5% CO2 atmosphere at 37 C. After culture, cells were separated from the culture medium by centrifugation for 10 min at 400 x g at room temperature.

To study the effects of IGF-I on apoptosis or cytokine production, granulocytes were cultured in the absence or presence of 1.3, 6.5, or 40 nM IGF-I. In all experiments, IGF-I was added directly at the start of the culture period. Assessment of apoptosis by microscopic examination and DNA fragmentation assays was performed after a 5-h culture period. Assessment of apoptosis by measuring relocation of phosphatidylserine using Annexin V binding assays was done after a 3-h culture period because relocation of phosphatidylserine is an early marker of apoptosis and precedes morphological changes and DNA fragmentation. When the effects of IGF-I on cytokine secretion by granulocytes or PBMC were assessed, cells were cultured for 18 h and stimuli (GM-CSF or LPS) were added 1 h after addition of IGF-I.

Radioligand binding assays
Recombinant human IGF-I was radioiodinated to a specific activity of 20–60 megabecquerels/nmol using the chloramine-T method. Competition binding assays were carried out as described earlier (14), and the number of receptors per cell and their affinity for IGF-I were calculated by linear subtraction analysis (15).

Morphological assessment of apoptosis
Cells attached to poly-L-lysine coated slides, were fixed and stained with Diff-Quick according to the manufacturer’s procedure (Dade Behring S.A., Brussels, Belgium). The proportion of apoptotic cells was determined by light microscopy; apoptotic granulocytes were defined as cells containing pyknotic nuclei (see Fig. 2AGo; 16). Granulocytes from five different donors were tested and all incubations were performed in 5-fold.



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  		Figure 2. Apoptosis was assessed by microscopic analysis after a 5-h culture period. Photographs of cells that were cultured in the absence (A) or in the presence of 40 nM IGF-I (B) are shown. The arrowsheads indicate apoptotic granulocytes with nuclear condensation. Magnification, 350x. C, Inhibition of apoptosis by IGF-I. The data represent mean inhibition ± SEM (n = 5; all females). The percentage of apoptotic cells increased from 0.20 ± 0.18% to 3.32 ± 0.57% in the absence of IGF-I. Significance is indicated by a (P < 0.05) and b (P < 0.01).

 
Annexin V binding assay
Expression of phosphatidylserine in the outer leaflet of the plasma membrane (an early marker of apoptosis) was detected by binding of fluorescein isothiocyanate (FITC)-conjugated annexin V using the annexin V-FITC kit from Bender Medsystems (Vienna, Austria). Viable cells were distinguished from necrotic cells by simultaneous staining with propidium iodide (PI). Cells stained positive for annexing V and negative for PI were considered apoptotic. Stained cells were analyzed on a fluorescence-activated cell sorter Vantage flow cytometer. For each sample forward light scatter, side scatter, green fluorescence (FITC), red fluorescence (PI) of 10,000 cells were acquired. The spectral overlap of the different fluorescence emission spectra was corrected by electronic compensation. Debris and remaining erythrocytes were excluded by gating on basis of forward and side scatter. Data analysis was performed using Winmdi software (URL: http://facs.scripps.edu). Granulocytes from five different donors were tested, and all incubations were performed in 5-fold.

DNA fragmentation assay
Cells attached to poly-L-lysine coated slides were fixed in freshly prepared 4% methanol-free formaldehyde solution in PBS (pH 7.4) for 10 min at room temperature. Subsequently, fragmented DNA was detected by labeling DNA ends using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphospate (dUTP) nick end-labeling (TUNEL) kit from Promega Corp. (Madison, WI). The percentage of labeled cells was determined by fluorescent microscopic analysis instead of flow cytometry to distinguish DNA end-labeling from aspecific binding of FITC-labeled dUTP to eosinophilic granules (17). Consequently, cells with cytoplamic staining only were considered to be normal. DNA fragmentation was quantitated by assessing the fraction of cells with nuclear staining as a percentage of all granulocytes. Granulocytes from six different donors were tested and all incubations were performed in triplicate.

Cytokine assays
Culture media were frozen and stored at -20 C until use. The levels of IL-1ß, IL-6, TNF{alpha}, or IL-8 were quantified by ELISAs using commercial antibody pairs (Cytosets) from Biosource International (Nivelles, Belgium). ELISAs were carried out according to the manufacturer’s protocol using the following antibody concentrations: TNF{alpha} coating antibodies (68B2B3 and 68B6A3), total concentration 1.0 µg/ml; TNF{alpha} detection antibody (68B3C5), 0.25 µg/ml; IL-6 coating antibody (677B6A2), 1.0 µg/ml; IL-6 detection antibody (505E23C7), 0.2 µg/ml; IL-8 coating antibody (893A6G8), 0.5 µg/ml; IL-8 detection antibody (790A28G2), 0.05 µg/ml; IL-1ß coating antibody (508A7G8), 1 µg/ml; IL-1ß detection antibody (508A3H12), 0.5 µg/ml. The sensitivities of the ELISAs for TNF{alpha}, IL-1ß, IL-6, and IL-8 were 10, 10, 4, and 3 pg/ml, respectively.

Statistical analysis
Values are presented as the mean ± SEM. To determine statistical significance, the unpaired t test with Bonferroni’s correction was used. Means were considered to be statistically different at P values < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I receptor expression
IGF-I receptor expression on granulocytes was studied by radioligand binding assays. Figure 1Go shows that 83% of the total binding of 125I-IGF-I can be displaced by rhIGF-I. The specificity of the IGF-I-binding sites was confirmed by competition with different concentrations of insulin that competed with 125I-IGF-I for the binding sites with a 2,000 times lower potency than IGF-I. Preincubation of cells with a monoclonal antibody against the IGF-I receptor ({alpha}IR3) resulted in a 71% inhibition of specific binding, indicating that IGF-I binding sites represent bona fide IGF-I receptors. Although IGF-I receptors were detected on granulocytes from all seven donors tested, the number of receptors per cell (1390 ± 467) and the dissociation constant (Kd) (2.3 ± 0.9 nM) were highly variable, and ranged from 246-3739 receptors/cell and 0.2–5.6 nM, respectively. Our observation that granulocytes express IGF-I receptors is in accordance with the finding of others that {alpha}IR3 blocks the stimulating effects of IGF-I on the oxidative burst (11, 12).



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  		Figure 1. Competition binding assay for 125I-IGF-I on human granulocytes. Suspensions of granulocytes were incubated with 0.24 nM 125I-IGF-I and increasing concentrations of unlabeled IGF-I ({diamond}) or insulin ({blacksquare}). Binding of 125I-IGF-I was also assessed in the presence of 10 µg/ml {alpha}IR3 ({square}). Data points are the mean ± SEM of triplicate incubations and representative of seven independent experiments (2 males; 5 females).

 
Inhibition of apoptosis by IGF-I
The level of apoptosis as assessed by microscopical analysis in freshly isolated granulocytes was 0.2 ± 0.4%. The effects of IGF-I on spontaneous apoptosis of granulocytes was measured after a 5-h culture period in serum-free medium, because overnight cultures in serum-free medium resulted in a substantial loss (50–70%) of intact granulocytes as assessed by microscopic analysis of Diff-Quick-stained cells. The loss of structural integrity did not allow to differentiate between normal, apoptotic and necrotic cells. The micrographs in Fig. 2Go show normal cells with bilobed or multilobed nuclei and apoptotic cells with pyknotic nuclei after a 5-h culture period in the absence (A) or presence of IGF-I (B). It appears that the fraction of apoptotic cells is smaller in the presence of IGF-I. Figure 2CGo reveals that 6.5 and 40 nM IGF-I significantly inhibit the onset of granulocyte apoptosis by 34 and 37%, respectively. A separate and independent determination of apoptosis was carried out by measuring the presence of phosphatidylserine on the outside of the plasma membrane using FITC-labeled annexin V. Figure 3Go, A and B, shows a marked reduction in the proportion of apoptotic cells (annexin V positive/PI negative; see lower right quadrants) due to IGF-I treatment. The number of necrotic cells (upper left quandrants) was not affected. The average reduction in the percentage of cells undergoing apoptosis in the presence of 40 nM IGF-I was 43% (Fig. 3CGo). Because apoptosis is usually characterized by the generation of DNA fragments through the action of endogenous endonucleases, we assessed the effects of IGF-I on DNA fragmentation using the TUNEL assay. Brightly stained cells in Fig. 4Go, A and B, represent TUNEL-positive cells. It appears that cells cultured in the presence of 6.5 nM IGF-I (Fig. 4BGo) display less DNA fragmentation. Quantitative analysis revealed that a concentration of 6.5 nM IGF-I significantly inhibited DNA fragmentation by 45% (Fig. 4CGo), indicating that the antiapoptotic effect of IGF-I as measured by morphological analysis and phosphatidylserine relocation concurs with an inhibition of DNA fragmentation.



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  		Figure 3. Apoptosis was assessed by flow cytometric analysis of cells that were simultaneously stained with annexin V and PI. Dotplots of cells cultured for 3 h in the absence (A) or presence (B) of 40 nM IGF-I are shown. The proportion of cells in each quadrant is given as the percentage of forward- and side-scatter gated cells. Annexin V-positive/PI-negative cells (lower right quadrant) are apoptotic. C, Inhibition of apoptosis by IGF-I. The data represent mean inhibition ± SEM (n = 5; all females). The percentage of apoptotic cells in freshly isolated granulocytes was 2.30 ± 0.15% which increased to 10.6 ± 4.1% after a 3-h culture period without IGF-I. Significance is indicated by a (P < 0.05).

 


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  		Figure 4. Inhibition by IGF-I of DNA fragmentation. Nuclei were stained with PI (red fluorescence) and TUNEL was performed with dUTP-FITC (green fluorescence). To assess the fraction of apoptotic cells by TUNEL assay, samples were illuminated with UV light to visualize TUNEL-positive nuclei using filter settings for FITC staining (420 nm). Photographs were taken with a Carl Zeiss (Zaventem, Belgium) Axiophot fluorescence microscope using a Tmax (Kodak, Rochester, NY) black and white film. Due to the strong staining with PI, all nucleated cells are visible using filter settings for FITC staining and appear as weakly stained cells. FITC-labeled cells displaying DNA fragmentation are brightly stained (see arrows). The photographs (magnification, 350x) show cells from samples that were taken after a 5-h culture period in the absence (A) or presence (B) of 6.5 nM IGF-I. C, Inhibition of DNA fragmentation by IGF-I. The data represent mean inhibition ± SEM (n = 6; 2 males; 4 females). Significance is indicated by a (P < 0.05). The percentage of cells in which DNA fragmentation was detected increased from 0.60 ± 0.45% to 12.0 ± 3.1% during the 5-h culture period in the absence of IGF-I.

 
IGF-I does not affect cytokine secretion by granulocytes
Granulocytes secrete substantial levels of IL-8 and low levels of other cytokines such as TNF{alpha}, IL-6, and IL-1ß (1). To evaluate whether granulocyte-derived cytokines could mediate the effects of IGF-I on apoptosis, we measured the effects of IGF-I on cytokine secretion by granulocytes from ten donors. As shown in Table 1Go, granulocytes from all donors secreted significant amounts of IL-8, which were not influenced by IGF-I. Small amounts of TNF{alpha} were secreted by granulocytes from four out of ten donors, whereas five donors secreted significant levels of IL-6. Again, cytokine secretion was not affected by IGF-I. IL-1ß levels in the culture medium, however, were below the detection limit of 10 pg/ml.


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  		Table 1. Effects of IGF-I on cytokine secretion by granulocytes and PBMC1

 
To further evaluate whether IGF-I may influence leukocyte recruitment to sites of inflammation through modulation of IL-8 secretion, we investigated the effects of IGF-I on IL-8 secretion by monocytes and activated granulocytes. Table 1Go shows that IGF-I does not influence the secretion of IL-8 by granulocytes stimulated with a suboptimal dose of GM-CSF (10 ng/ml). On the other hand, we found that 40 nM IGF-I significantly enhances basal secretion of IL-8 by PBMC and that 6.5 and 40 nM augments IL-8 secretion by LPS-stimulated PBMC. In each experiment, the effects of IGF-I and LPS were synergistic. We also observed positive effects of IGF-I on TNF{alpha} and IL-6 secretion by LPS-stimulated PBMC (Table 1Go). These observations are consistent with the findings of other groups showing positive effects of IGF-I on the secretion of TNF{alpha} by monocytes (8) and IL-6 by PBMC (10). Taken together, these results demonstrate that although IGF-I has stimulatory effects on the secretion of inflammatory cytokines by PBMC, the secretion of these cytokines by granulocytes is not affected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although granulocytes are terminally differentiated leukocytes that are constitutively committed to apoptosis, their life span is regulated by cytokines and other inflammatory mediators through acceleration or inhibition of apoptosis. For instance, exposure to IL-10 stimulates apoptosis (18), whereas LPS, GM-CSF, and IL-8 inhibit apoptosis (19, 20, 21, 22). IGF-I is an ubiquitous survival factor that inhibits apoptosis in many cell types. Because IGF-I inhibits apoptosis in promyeloid HL-60 cells and promotes their differentiation toward granulocytes (7), we evaluated the effects of physiological concentrations of IGF-I on spontaneous apoptosis of freshly isolated peripheral blood granulocytes. The presence of IGF-I receptors with a Kd of 2.3 ± 0.9 nM indicates that physiological effects of IGF-I are possible. Furthermore, we show that IGF-I inhibits spontaneous apoptosis of granulocytes as assessed by three different assays for apoptosis: microscopical analysis, Annexin V binding, and DNA fragmentation assay. The magnitude of the inhibition by IGF-I is comparable to the effects of GM-CSF and IL-8 as observed by Klein et al. (21). Interestingly, PI3K and extracellular regulated kinase, which both mediate the inhibiting effects of GM-CSF and IL-8 on apoptosis (21), are also targets for IGF-I receptor signaling (23). It remains to be established whether IGF-I inhibits granulocyte apoptosis through PI3K as observed for leukemic myeloid progenitors (7).

As granulocytes play an important role in inflammation, most studies on cytokine production by these cells have focused on inflammatory cytokines. It has been shown that granulocytes produce large amounts of IL-8 and small amounts of other inflammatory cytokines such as IL-1ß, IL-6, and TNF{alpha} (1). Interestingly, these cytokines influence granulocyte apoptosis in several ways. IL-8 and IL-1ß inhibit neutrophil apoptosis (20, 21, 22, 24), whereas TNF{alpha} and IL-6, have been variously reported to accelerate or delay apoptosis, depending upon the experimental circumstances and the cell type studied (25, 26, 27, 28). To address possible autocrine of paracrine effects of granulocyte-derived cytokines on apoptosis, we evaluated the effects of IGF-I on cytokine secretion. We found that cytokine concentrations in the culture medium were markedly lower than those necessary for modulation of apoptosis in vitro (22, 26, 27), and that IGF-I does not affect the secretion of IL-8, IL-6, and TNF{alpha}. Therefore, we conclude that modulation of secretion of these cytokines by granulocytes is not instrumental to in vitro inhibition of apoptosis by IGF-I. Granulocytes were stimulated with a suboptimal concentration of GM-CSF to test the effects of IGF-I on activated granulocytes. GM-CSF stimulates degranulation, phagocytosis (29), and IL-8 secretion by neutrophils (1). The absence of any effect of IGF-I on cytokine secretion by unstimulated and GM-CSF-stimulated granulocytes suggests that these cells are not a target for modulation of IL-8 secretion by IGF-I. In contrast to granulocytes, PBMC respond to IGF-I by increasing basal IL-8 production (Table 1Go). These results suggest that IGF-I uses, at least in part, differential signal transduction pathways in these cell types. Distinct signal transduction pathways due to differential expression of insulin receptor substrate-1 or -2 have already been demonstrated in different developmental stages of T cells and HL-60 cells, respectively (30, 31).

Our data indicate that IGF-I may enhance granulocyte functions through effects on granulocyte longevity. Indeed, apoptosis has been associated with loss of neutrophil functions (32). IGF-I may act directly through IGF-I receptors on granulocytes, or indirectly through stimulating the secretion of IL-8 or other cytokines by monocytes or macrophages. Although IGF-I has been shown to be a chemotactic factor for T cells (33) and multiple myeloma cells (34), we were unable to detect any direct chemotactic activity of IGF-I on granulocytes (data not shown) as assessed by a single-filter assay (35). However, IGF-I may promote the recruitment of granulocytes to sites of inflammation by stimulating monocytes to increase IL-8 secretion. On the other hand, the primary regulator of IGF-I, GH, has been shown to inhibit formylmethionylleucylphenylalanine-induced chemotaxis of human neutrophils (36).

Circulating levels of IGF-I mainly depend on GH that stimulates IGF-I production in liver and other tissues. Although IGF-I production in liver is mainly controlled by GH, IGF-I expression in other tissues is also regulated by many other factors. IGF-I in circulation is mostly complexed to high-affinity IGF binding proteins (IGFBPs). Six IGFBPs have been well characterized (37), and they act as carrier proteins for IGF-I and IGF-II involved in transport and protection against proteolytic degradation. Importantly, IGFBPs are expressed in most tissues and have been shown to inhibit or enhance cellular effects of IGF-I. Thus, local, autocrine or paracrine, effects of IGF-I are not exclusively regulated through modulation of IGF-I or IGF-I receptor expression, but also through regulation of IGFBPs. Therefore, modulation of IL-8 secretion by monocytes and granulocyte apoptosis through IGF-I can be regulated at different levels. Obviously, these processes may be regulated through stimulation of circulating IGF-I levels by GH, nutritional status (37) or inflammatory cytokines such as IL-1ß and TNF{alpha} (38, 39). Alternatively, the effects of IGF-I may be regulated by modulation of local concentrations of free IGF-I via secretion of IGF-I and IGFBPs by leukocytes, or by modulation of IGF-R expression on target cells. Indeed, all elements of the IGF-I system are present in the immune system and can be regulated by immune specific stimuli. Most leukocyte subpopulations express IGF-I receptors (3, 4), and these receptors are regulated by immune specific activation pathways, such as signaling through the CD3 T cell receptor complex (40) and CD28 (6). Also the differential expression of IGF-I receptors on nonactivated and antigen-activated CD45R0+ T cells (41) and the developmental stage-dependent expression of IGF-I receptors on thymocytes (42) indicate that effects of IGF-I may be regulated through modulation of receptor expression. IGF-I is produced by macrophages (4), phytohemagglutinin-activated PBMC (43), and bone marrow stromal cells (44). Moreover, IGF-I production by macrophages is regulated by cytokines instead of GH (45, 46, 47), and prostaglandin E2 has been shown to stimulate IGF-I synthesis by osteoblasts (48). Taken together, these data suggest that the putative regulation of apoptosis by IGF-I may take place through regulation by cytokines of local IGF-I production by leukocytes. Alternatively, IGFBPs secreted by lymphocytes (43) or macrophages (49) may influence apoptosis by modulation of free IGF-I. Because the neutrophil proteases cathepsin G and elastase can cleave all six IGFBPs (50), it is intriguing to speculate that degranulation of granulocytes leads to degradation of IGFBPs resulting in increased concentrations of free IGF-I. Interestingly, the tumor suppressor gene product, p53, which induces apoptosis, has been shown to increase expression of IGFBP-3 (51) and to down-regulate IGF-I receptor expression (52, 53). We hypothesize that granulocyte longevity can be regulated through modulation of free IGF-I and IGF-I receptor expression. Further experiments are required to investigate the role of the IGF-I system in inflammatory reactions.

Whether the described effects of IGF-I provide new therapeutic means for treatment of inflammatory diseases, for instance by administration of IGFBPs to enhance granulocyte apoptosis and inhibit IL-8 production by monocytes, remains to be established. Beneficial effects of IGF-I have been observed in patients with ischemia. Because both IGF-I and the apoptosis inhibitor ZVAD-fmk inhibited renal apoptosis, inflammation and tissue injury in after reperfusion, it was concluded that inhibition of renal apoptosis prevents inflammatory reactions (54). In burned children, treatment with IGF-I and IGFBP-3 resulted in an inhibition of the acute phase response and reduction of serum levels of TNF{alpha} and IL-1ß (55). These results contradict in vitro experiments showing that IGF-I enhances the secretion of proinflammatory cytokines (8). However, the IGF-I system is very complex, because it operates in autocrine, paracrine and endocrine fashions and involves stimulating or inhibiting effects of IGFBPs that are present in circulation and produced by many tissues (23). Therefore, the way of IGF-I administration (with or without IGFBPs, systemically or locally) and the tissues involved may determine the outcome of IGF-I treatment.


   Acknowledgments
 
We are grateful to Eli Lilly \|[amp ]\| Co. for providing rhIGF-I. We thank Peggy Verdood and Isabelle Vandriessche for their technical assistance, and Dr. R. Hooghe for critical discussion of the manuscript.


   Footnotes
 
This research has been funded by the Flemish Government (GOA 97-02-4), the Fund for Scientific Research-Flanders (F.W.O. G0167.98N), and institutional grants from the V.U.B.

Abbreviations: dUTP, Deoxyuridine triphosphate; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-monocyte colony stimulating factor; {alpha}IR3, IGF-I receptor; IGFBPs, IGF binding proteins; Kd, dissociation constant; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; PI, propidium iodide; rh, recombinant human; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Received September 12, 2001.

Accepted for publication December 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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