Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility

Neuropeptides(1996) 30 (6), 583-595 © PearsonProfessionalLtd 1996

Pituitary adenylate cyclaseactivating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility E. Garrido ~, M. Delgado ~, C. Martinez ~, R.P, Gomariz ~, M. De la Fuente 2 ~Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense de Madrid, 28040 Madrid 2Departamento de Fisiologia Animal, Facultad de Biologia, Universidad Complutense de Madrid, 28040 Madrid, Spain

Summary The effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) in a concentration range from 10-13 to 104 M were studied, in vitro, on two functions of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that PACAP38 raised the adherence of the two cell types, increased the mobility of macrophages and decreased the mobility of lymphocytes. The maximal effects were observed at 10-l° M in macrophages and at 10-9 M in lymphocytes. Moreover, incubation with increasing concentrations of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, resulted in a progressive enhancement of adherence and chemotaxis of both macrophages and lymphocytes. In contrast, retinal, a PKC inhibitor, significantly decreased these capacities. Incubation of macrophages with both PMA and PACAP38 did not have a synergistic effect on chemotaxis and adherence whereas, with lymphocytes, adherence was increased and chemotaxis was partially decreased. On the other hand, incubation with forskolin (an enhancer of intracellular cyclic AMP [cAMP] levels) caused inhibition and stimulation of chemotaxis and adherence, respectively, in both cell types. PACAP38 prevented the inhibitory effect of forskolin on chemotaxis of macrophages but not of lymphocytes, whereas the simultaneous presence of PACAP38 and forskolin was synergistic for adherence of both peritoneal cells. In addition, PACAP38 was chernoattractant for macrophages but not for lymphocytes. Furthermore, a VtP receptor antagonist was able to partially rew~rse the modulatory effects of PACAP38 on lymphocytes, but not on macrophages. These data suggest that PACAP38 exerts its action through the binding to type I PACAP receptors and PKC activation in macrophages and through the elevation of intracellular cAMP levels by binding to type II PACAP receptors in lymphocytes. The present work reveals an additional link between neuropeptides and the immune system and suggests that the peptide PACAP modulates the immunological function of macrophages and lymphocytes.

INTRODUCTION

It has been established that there is a functional relationship between the nervous and the immune system and that Received 17 September 1996 Accepted 23 September 1996 Correspondence to: Rosa P. Gomariz, Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense de Madrid, 28040 Madrid, Spain. Tel: +34 (1) 394 4971. Fax: +34 (1) 394 4981.

neuropeptides play a key role linking the nervous and the immune systems through specific membrane receptors. 1 The pituitary adenylate cyclase-activating peptide (PACAP) is a recently found neuropeptide of the VIP/glucagon/secretin family of peptides? The N-terminal sequence of PACAP shows a 68% homolog3r with the vasoactive intestinal peptide (VIP)? PACAP is present in two amidated forms, PACAP27 and PACAP38 (PACAP38 being the predominant form) and the PACAP38/PACAP27 583

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Garrido et al

ratio varies among tissues, especially in the central nervous system, adrenal and testis, suggesting different processing in different tissues. Recently, PACAP immunoreactivity has been described in central and peripheral lymphoid organs 4 and PACAP receptors seem to be present in splenic lymphocytes? However, there is a scarcity of reported effects of PACAP38 on immune cells. It inhibits the mitogen-stimulated proliferation of murine splenocytes5 and enhances the phagocytic function of mouse macrophages. 6 Moreover, we have recently shown the inhibition of spontaneous mobility and chemotaxis of murine thymocytes and splenic lymphocytesz and the inhibition of thymocyte apoptosis 8 by PACAP27 and PACAP38. Two PACAP receptor types have been described: the type I receptor (selective receptor) which binds PACAP27, PACAP38 with higher affinity than VIP 9,~°and the type II receptor (non-selective) which binds both PACAPs and VIP with similar affinities? These belong to the secretin family of receptors which are coupled to G protein and activate adenylate-cyclase.11,~2Further, the PACAP receptor type I may interact with the G protein that mediates the activation of phospholipase C. ~3 Adherence and mobility are crucial for the immune response and the first mechanisms involved in the inflammatory response. 14,15 These functions are shared by the two principal types of immune cells, i.e. lymphocytes and phagocytes? 6 Since there are no data available on the biological effect of PACAP on adherence and mobility of lymphocytes and phagocytic cells, and these two kinds of cells are found together and in similar amounts in murine peritoneum, we studied the in vitro effects of PACAP38 in the range 10-~3-10-6 M on those functions in peritoneal cell populations. Recent resuks from our laboratory show the existence of receptor type I of PACAP in rat peritoneal macrophages and type II receptor in both peritoneal populations: lymphocytes and macrophages. Therefore, the immunological action of PACAP38 seems to be exerted through these specific receptors in peritoneal cells. There is a considerable homology between VIP and PACAP with binding sites shared by both neuropeptides and VIP seems to act through a different second messenger in murine peritoneal macrophages and lymphocytes? 7,1a Therefore, another aim of this study is to investigate the type of receptor involved and the intracellular signal pathways in both types of immune cells. MATERIALS AND METHODS Chemical reagents

PACAP38 was purchased from Novabiochem (Switzerland). VIP and VIP antagonist [N-Ac-Tyr1, D-PheZ]-GRF (1-29) Neuropeptides (1996) 30(6), 583-595

amide were obtained from Cambridge Research BioChemicals (Wilmingtong, DE). Phorbol myristate acetate (PMA), forskolin, N-formyl-Met-Leu-Phe (FMLP), and retinal were purchased from Sigma (St Louis, MO), and transparent nitrocellulose filters (3 gm pore diameter) for chemotaxis from Millipore (Bedford, MA). Hank's solution was prepared as follows: 5.5 mM glucose, 1 mM MgC12, 136 mM NaC1, 5 mM KC1, 1 mM CaC12, 0.8 mM MgH2PO4, 0.5 mM KH2PO4, 0.4 mM Na2HPO 4 and 4 mM NaHCO3, adjusted to pH 7.4. Incubations were performed at 37°C, with 5% CO 2 and humidified atmosphere. The cells were counted in Neubauer chambers. Animals

Male Wistar rats of 250-300 g body weight, 10 _+2 weeks old, were purchased from Iffa Credo (France), and maintained at constant temperature (22_+2°C) on a 12-h light/dark cycle. Animals were fed Sander Mus (Iffa Credo) and water ad libitum, Collection of peritoneal exudate cells

Each animal was killed by decapitation according to the guidelines by the European Community Council Directives 86/6091 EEC, the abdomen cleaned with 70% ethanol, the abdominal skin carefully dissected without opening the peritoneum and 10 ml of saline Hank's solution was injected intraperitoneally. Then, the abdomen was massaged and about 90% of the saline solution, containing lymphocytes and macrophages was extracted. Flow cytometric analysis showed a proportion of 40%, 50%, and 10% of macrophages, lymphocytes, and polymorphonuclear cells, respectively. Resting macrophages, determined by morphology and nonspecific esterase staining, were counted in Neubauer chambers, and were adjusted in the same medium at 5 x 105 macrophages/ml or 5 x 105 lymphocytes/ml. Cellular viability, routinely determined before and after each experiment using the test of trypan blue exclusion, was in all cases higher than 95%. Adherence assay

The adherence assay was carried out as previously described. 19 Briefly, aliquots of 200 gl of adjusted macrophage or lymphocyte suspensions were transferred to Eppendorf tubes, the adherence which resembles that of tissuesY 20 gl PACAP38 were added to reach a fmal concentration from 10-a M to 10-13 M. 20~11of Hank's solution was added in the control samples. Adherence assays were performed at 5, 10, 30, and 60 min and, after a gentle agitation of cell suspensions, 10 gl were taken and the number of nonadherent macrophages and lymphocytes/ml © Pearson Professional Ltd 1996

PACAP modulates adherence and mobility 585

was counted in Neubauer chambers. The adherence index (AI) was calculated according to the following equation, in which cells represent macrophages or lymphocytes: [.AI=I - (cells/ml supernatant)/(cells/ml Ol~Lginalsample)] x 100 In some experiments, FMLP at a concentration of 10-8 M was added as a negative controlY VIP (10-9-10 -1° M) was used as a control for neuropeptide-modulation of adherence capacity in peritoneal macrophages and lymphocytes. Mobility assay

Mobility assays were performed following a modification TM of the original technique, 21which consists basically of the use of chambers comprising two compartments separated by a nitrocellulose filter with a pore diameter of 3 gm. Aliquots of 300 gl of peritoneal suspension (adjusted macrophages or lymphocytes) were deposited in the upper compartment of the Boyden chambers with 30 gl of PACAP38, at a final concentration ranging from 10-6 to 10-" M, or 30 pl of Hanks solution (controls). Aliquots of 400 gl of Hank's solution (spontaneous mobility) or FMLP at a concentration of 10-8 M (chemotax3s) were put into the lower compartment. The chambers were incubated for 3 h at 37°C, after which the filters were fixed, stained, and mounted using histological methods. The mobility index (spontaneous mobility and chemotaxis) was determined by counting in an opticol microscope (immersion objective) the total number of macrophages or lymphocytes in one third of the lower face of the filter. In some experiments, VIP (10-9-10 -1° M) was used as a control for neuropeptide-modulation of chemotaxis capacity in peritoneal macrophages and lymphocytes. Chemoattractant activity

The assay to study the chemoattractant activity of PACAP38 was carried out as described above for the mobility assay, but in this case the neuropeptide was deposited in the lower compartment of the chamber at concentrations ranging from 10-z M to 10-11 M. In other samples, FMLP, a well-known chemoattractant for macrophages 22 and lymphocytes 23 at a concentration of 10-s M, and Hank's solution were added in the lower compartment as a positive and negative control, respectively. The chemoattractant index was estimated counting the total number of macrophages or lymphocytes migrating through the filter in eight powered fields in duplicate. In some experiments, VIP (10-9-10 -1° M) was used as a control for neuropeptide-modulation of chemoattractant activity in peritoneal macrophages and lymphocytes. © Pearson Professional Ltd 1996

Action mechanisms of the modulatory effect of PACAP38 on adherence and chemotaxis

When indicated, in order to determine the intracellular signal pathways involved in the modulation of adherence and chemotaxis of peritoneal macrophages and lymphocytes by PACAP38, we measured the effect of phorbol myristate acetate (PMA) (a protein kinase C [PKC] activator), of forskolin (an enhancer of cyclic AMP [cAMP1 levels24), and of retinal (an inhibitor of PKC activity25) on these phagocytic functions. Adherence and chemotaxis assays were evaluated as indicated above using PACAP38 at a final concentration of 10-1° M. PMA (0.5-50 ng/ml), retinal (10-6-10 a M), or forskolin (10-5-10 -7 M) were added in the presence or absence of neuropeptide. Effect of VIP receptor antagonist on adherence and chemotaxis

To determine the specificity of PACAP38 effects and the type of receptor implicated, the effect of the VIP receptor antagonist [N-Ac-Tyr1, D-Phe2]-GRF (1-29) NH2, on adherence and mobility was evaluated as described above using PACAP38 and VIP at a final concentration of 10-1° M or 10-9 M, respectively. VIP antagonist (10-5-10 -8 M) was added in the presence or absence of neuropeptides. Statistical analysis

All values are expressed as the mean _+SD of the number of experiments performed in duplicate, as indicated in the corresponding tables and figures. The data were evaluated statistically by single factor ANOVA of repeated measures for paired observations of parametric data followed by Scheffe's F-test between two groups, with P < 0.05 as the minimum level of significance. RESULTS PACAP38 stimulates the adherence capacity of rat peritoneal macrophages and lymphocytes

Fig. 1 shows the adherence index of peritoneal macrophages incubated wffh PACAP38 at 5, 10, 30, or 60 min. PACAP38 significantly stimulated the adherence capacity of macrophages at concentrations between 10-6 M and 10-12 M, at all incubation times, compared with controls. The highest stimulation was produced by 10-1° M of PACAP38. In addition, our results show that PACAP38 induced a significant increase in peritoneal lymphocyte adherence (concentration ranging from 10-6-10 -12 M) with a maximum effect at 10-1°-10-9 M of PACAP38, already significant at the shortest time of 5 mill (Table 1). To exclude the possibility of the nonspecific effect of covering the surface of the tubes with Neuropeptides (1996) 30(6), 583-595

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Garrido et al

ll0

,

I~P-

5 min 10 min 30 min 60 min

100 90 80 70 60

<

50 40 30

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I

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13

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12

I

11

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I

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[

I

10

9

8

7

6

[PACAP381 (-log M)

Fig. 1. Effect of PACAP38 on the adherence index of rat peritoneal macrophages. Peritoneal suspensions were incubated with different concentrations of PACAP38 or Hank's solution (controls) for the indicated time. The adherence index was determined as described in Materials and Methods. The results represent the mean +- SD of eight experiments performed in duplicate. The adherence index of peritoneal suspension treated with 10 -6 M VIP was: 60+-4 at 5 min, 66+-5 at 10 min, 82+-5 at 30 min, and 92+-4 at 60 min; and with 10 -8 M FMLP was: 33+-4 at 5 min, 43+-4 at 10 min, 51+-7 at 20 min, and 59+-9 at 60 min. Significant differences from untreated controls at each corresponding time are marked as follows: **P< 0.01, ***P< 0.001.

positive charges, a group of control tubes was pretreated with PACAP38 (10-l° M). The neuropeptide was removed by washing and the tubes were incubated with cells. No significant differences were found in the adherence index compared to controls: 41_+3 (5 re_in), 52+4 (10 min), 64+4 (30 min), and 70+6 (60 rain) in lymphocytes; 43+3 (5 rain), 55+2 (10 min), 64+8 (30 min), and 74+7 (60 min) in macrophages. In order to assess the results obtained with PACAP38, a positive control with VIP (10-9 M) and a negative control with FMLP (10 -8 M) were included in the adherence assay. VIP showed a significant increase (P<0.O01) in the adherence indexes of macrophages and lymphocytes compared with these control samples (Fig. 1 and Table 1). FMLP significantly decreased (P <0.001) adherence indices of lymphocytes and macrophages compared to those of control samples (Fig.1 and Table 1). Since the highest stimulation of adherence capacity was obtained with 10-1° M PACAP38 at 10 min of incubation, these optimal conditions were selected to further study the stimulatory effect and action mechanism of PACAP38 on the adherence of macrophages and lymphocytes. Modulatory effect of PACAP38 on mobility capacity in rat peritoneal macrophages and lymphocytes

After determining the stimulatory effect of PACAP38 on adherence, we studied its effect on the mobility of macrophages and lymphocytes. PACAP38 significantly stimulated the spontaneous mobility index of macrophages between 10-6 M and 10-12 M, the highest stimulation being produced by 10-l° M (Table 2). However, opposite results were found for lymphocytes with a similar

Table I The effect of PACAP38 on the adherence index for the substrate of peritoneal lymphocytes. Concentration Duration of incubation

Controls

10 -13 M

10-12 M

10 -11 M

10 -10 M

10-9 M

10 8 M

10 -z M

10-8 M

5 min 10 min 30 min 60 min

38 47 60 65

39 51 61 67

49 59 71 73

55 66 76 78

71 80 87 90

67 76 83 89

56 68 80 81

56 66 76 79

51 58 74 76

+2 +2 -+ 2 +2

-+ 5 +- 5 +8 +2

+ 4*** + 4*** _+5*** _+6*

+ 5*** + 5*** + 4*** _+6***

+- 6*** +_7*** +- 4*** :t: 4***

+_4*** + 4*** + 4*** + 4***

_+6*** + 6*** + 5*** +- 4***

+ 7*** + 3*** + 5*** +- 7**

+ + + +

6*** 6*** 4*** 3***

Peritoneal suspensions were incubated with different concentrations of PACAP38 or Hank's solution (controls) for the indicated time. The adherence index was determined as described in Materials and Methods. The results represent the mean + SD of eight experiments performed in duplicate. Significant differences from untreated controls at corresponding times are marked as follows: *P < 0.05,

**P< 0,01 ,***P< 0.001. The adherence index of peritoneal suspension treated with VIP (104 M) was: 58+_5 at 5 min, 64+-6 at 10 min, 71+-5 at 30 min, and 80+-6 at 60 min; and with FMLP (10 -8 M): 32+3 (5 min), 44+-4 (10 min), 53+-5 (20 min), and 60+7 (60 min).

Neuropeptides (1996) 30(6), 583-595

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PACAP modulates adherence and mobility 587

Table 2 The effect of PACAP38 on the spontaneous mobility index of peritoneal lymphocytes and macrophages. Concentration Controls

10-~3 M

10-~2 M

10"M

10~0M

10-9M

10-8M

10-7M

10-6M

Lymphocytes

392 + 49

372 _+44

331 + 47*

315 + 29**

264 + 35***

252 _+31"**

285 _+29***

322_+ 28**

Macrophages

370 _+27

392 _+25

427 + 22*** 484_+ 31"**

623 + 35***

598 4- 36***

561 + 5t***

459 + 24 .... 432 + 52**

336 + 33*

Cells were incubated with different concentrations of PACAP38 for 3h at 37°C in the upper chamber, and Hank's solution was pllaced in the lower chamber. In the case of controls, cells were incubated with buffer only. Spontaneous mobility index was determined as described in Materials and Methods. Each value is the mean 4- SD of eight experiments performed in duplicate. *P < 0.05,**P < 0.01 ,*** P < 0.001 with respect to controls.

range of significant concentrations, but the highest inhibition was produced by 10-9 M of PACAP38 (Table 2). The opposite effects of PACAP38 on the chemotaxis capacity of lymphocytes and macrophages are shown in Fig. 2. PACAP38 at concentrations ranging from 10 -~ M to 10-12 M caused a significant stimulation of macrophage chemotaxis. Again, the most effective concentration was 10 -1° M. PACAP38 also produced a significant inhibition of lymphocyte chemotaxis concentrations of from 10 -6 M to 10 -~2 M. Maximum inhibition was obtained using l0 -9 M PACAP38. VIP (10 -1° M or 10 -9 M) was used as a positive control to assess the results of PACAP38. VIP significantly stimulated (P<0.001) the chemotaxis index of macrophages (Fig. 2): 838_+91 by 10 -1° M and 1047+109 by 10 -9 M. The chemotaxis of peritoneal lymphocytes decreased (P<0.001) by incubation with VIP (Fig. 2): 428+48 by 10 -1° M and 398_+36 by 10 -9 M. The results obtained on the chemoattractant capacity of PACAP38 are shown in Fig. 3. PACAP38, ranging in concentration from 10 -7 M to 10 -H M, was a chemoattractant for peritoneal macrophages but not for lymphocytes. However, a well-known chemoattractant for lymphocytes 23 and macrophages =, FMLP, at a concentration of 10 -8 M caused an enhancement of the chemoattractant index in both peritoneal lymphocytes and macrophages (Fig. 3). In addition, VIP (10 -9 M) controls showed a chemoattractant action in macrophages (583+45) but not in lymphocytes (387+39). Since the highest effects of PACAP38 on chemotaxis were found with 10 <° M, this concentration was selected for further studies on this function.

Intracellular signal pathways and specific receptors involved in the modulation of adherence and chemotaxis of peritoneal macrophages and lymphocytes by PACAP38 In order to study the mechanism underlying the modulatory effect of PACAP38 on peritoneal macrophages and © Pearson Professional Ltd 1996

1200

--1-- Macrophages ~Lymphocytes

***

¢~ 1000

..~ 800

O 600 400

200

I

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13

12

11

10

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6

[PACAP38] (-log M)

Fig. 2. Effect of PACAP38 on the chemotaxis index of peritoneal macrophages and lymphocytes. Cells were incubated with different concentrations of PACAP38 for 3 h at 37°C in the upper chamber, and FMLP (104 M) was placed in the lower chamber. The chemotaxis index was calculted as described in Materials and Methods. The results represent the mean + SD of eight experiments performed in duplicate. *P < 0.05, **P < 0.131, ***P < 0.001 (compared to control values). The chemotaxis index of peritoneal suspensions treated with VIP was: 838+91 by 10-lo M, and 1047+109 by 10 -9 M, in macrophages; and 428+48 by 10-9 M, and 398+36 by 10 -l° M, in lymphocytes.

lymphocytes, we measured the effect of PMA (an activator of PKC), of retinal (an inhibitor of PKC) and of forskolin (an enhancer of cAMP intracellular levels) on the adherence and chemotaxis indices of these immune cells, incubated in the presence or absence of PACAP38. Table 3 shows the effect of PMA (50-0.5 ng/ml) and retinal (10-6-10 -8 M) on the adherence capacity of peritoneal macrophages and lymphocytes incubated with 10-1° M PACAP38. When macrophages and lymphocytes Neuropeptides (1996) 30(6), 583-595

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1000

A

Macrophages

800 600 400

..= 200

o

0 800

il ill B

Lymphocytes

600 400 20O 0

C

FMLP

7

9

11

[PACAP381 (-log lv0

Fig. 3. Influence of PACAP38 and formyI-Met-Leu-Phe (FMLP) on the chemoattractant index of rat peritoneal macrophages (A) and lymphocytes (B). PACAP38, FMLP (104 M) or control medium was added in the lower chamber and then peritoneal suspension was applied in the upper chamber. The chemoattractant index was calculated as indicated in Materials and Methods. The results represent the mean + SD of eight experiments performed in duplicate. ***P < 0.001(compared to control values)

were incubated with PMA and retinal, an augmentation and a reduction, respectively, in the adherence index occurred compared to untreated samples (controls). The PMA-stimulation of the adherence index was slightly higher in macrophages than in lymphocytes. As stated above, 10-1° M PACAP38 caused a significant increase in the adherence index of both macrophages and lymphocytes. Simultaneous incubations with PMA and PACAP38 showed that this phorbol ester produced a moderate increase in the adherence capacity. This effect was higher in lymphocytes, with statistically significant differences at 50 and 5 ng/ml PMA compared with corresponding PACAP38-treated samples, while macrophages only showed a slight increase (Table 3). The inhibitory effect of retinal on adherence capacity was partially suppressed when retinal was incubated in the presence of PACAP38. Thus, PACAP38 reduced the inhibitory effect of retinal by 28-31% in macrophages and by 39-43% ill lymphocytes (Table 3). In the incubation with both retinal and PACAP38, retinal produced a higher inhibition ill Neuropeptides (1996) 30(6), 583-595

macrophages than in lymphocytes in adherence capacity at all concentrations assayed (retinal reduced the PACAP38-induced adherence by: 28% for 10-~ M, 20% for 10-7 M, 12% for 10-8 M retinal in macrophages; and 23% for 10-6 M, 14% for 10-ZM,< 1%for 10-8 M retinalin lymphocytes) (Table 3). Table 4 shows the effect of different concentrations of forskolin on the adherence index of rat peritoneal macrophages and lymphocytes incubated in the presence of 10-~° M PACAP38. The incubation with increasing forskolin concentrations produced a progressively stimulatory effect in the adherence capacity of both types of peritoneal cells, with a higher effect in lymphocytes than in macrophages. A moderate increase in adherence index for both kinds of immune cells caused by forskolin was observed in the simultaneous incubation with forskolin and PACAP38. This stimulatory effect of forskolin on PACAP38-treated samples was higher in macrophages compared to lymphocytes. Thus, this effect of the neuropeptide-forskolin combinations was additive in macrophages at all forskolin concentrations tested, although no statistically significant differences were found between PACAP38-forskolin (10-5-10 -~ M) combinations in lymphocytes and the corresponding samples without neuropeptide (Table 4). Regarding chemotaxis in macrophages and lymphocytes, PMA caused a significant increase of the chemotaxis index and retinal produced a significant decrease compared to control samples (Table 3). The inhibitory effect of PACAP38 on lymphocyte mobility was prevented progressively by simultaneous incubation with increasing PMA concentrations (0.5-50 ng/ml) showing a dosedependent effect. In the presence of different concentrations of PMA, PACAP38 reduced the PMA-induced chemotaxis index of lymphocytes by 31-32%. These values were similar to the inhibition percentage (30%) observed for PACAP38 compared to control samples. When lymphocytes were incubated simultaneously with retinal and PACAP38, the highest inhibition of the chemotaxis index was found, showing a significant additive effect in PACAP38-retinal (10-6-10-z M) combinations compared to the corresponding neuropeptide-treated samples, without retinal (Table 3). When macrophages were incubated with both PMA and PACAP38, chemotaxis was not augmented when compared to PACAP38 alone (Table 3). Furthermore, the reduction of chemotaxis by retinal was partially suppressed when macrophages were incubated in the presence of PACAP38. As shown in Table 4, incubation with increasing forskolin concentrations produced a progressively inhibitory effect on the chemotaxis activity in both types of peritoneal cells. PACAP38 prevented this dose-dependent inhibitory effect of forskolin on macrophages. Statistically significant differences were found, at all concentrations © Pearson Professional Ltd 1996

PACAP modulates adherence and mobility 589

Table 3 The effect of PMA and retinal on adherence and chemotaxis of rat peritoneal macrophages and lymphocytes. Adherence a

Chemotaxis ~

Treatment Macrophages Controls PACAP38 PMA 50 ng/ml PMA 5 ng/ml PMA 0.5 ng/ml PACAP38 + PMA 50 ng/ml PACAP38 + PMA 5 ng/ml PACAP38 + PMA 0.5 ng/ml Retinal 104 M Retinal 10-7 M Retinal 104 M PACAP38 + retinal 10-6 M PACAP38 + retinal 10-7 M PACAP38 + retinal 104 M

51 76 86 72 64 84 81 77 38 42 48 55 61 67

Lymphocytes

±3 ± 6* ± 5* ± 5* ± 6* ± 6 *9 ± 4* ± 4* ± 6* _+7* ±4 ± 5{ ± 6"~ ± 5*9

47 79 60 56 51 86 84 78 37 41 45 61 68 79

Macrophages

±3 ± 4* ± 4* ± 4* ±4 ± 3*9 ± 2*§ ± 3* ± 4* ± 3* ±4 ± 4*9 _+3*9 + 4*

640 1145 997 864 827 1166 1147 1090 429 513 569 726 849 929

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Lymphocytes

38 112" 96* 107* 78* 100" 86* 98* 48* 38* 40* 54*9 52 *9 84*9

641 451 918 827 749 634 562 517 423 500 578 269 299 411

_+46 ± 61" ± 89* ± 86* ± 87* ± 639 ± 57*9 ± 68* ± 63* ± 33* ± 55 ± 33*9 ± 42"{ ± 53*

Peritoneal suspensions were incubated with 10-10 M PACAP38 or Hank's solution (controls) in the presence or absence of different concentrations of PMA or retinal. The adherence index and chemotaxis index were determined as described in Materials and Methods. a: The adherence index was determined after 10 min of incubation, b: The cells were incubated with PACAP38, PMA and/or retinal for 3 h in the upper chamber. FMLP (104 M) was placed in the lower chamber. Each value is the mean ± SD of six experiments performed in duplicate. *P < 0.05 (compared to controls), ~P < 0.05 (compared to corresponding PACAP38-treated samples, without PMA or retinal)

Table 4 The effect of forskolin on the adherence and chemotaxis index of rat peritoneal macrophages and lymphocytes. Adherence a Treatment FK 104 M FK 10_6 M FK 10q M Controls PACAP38 PACAP38 + FK 10-5 M PACAP38 + FK 104M PACAP38 + FK 10_7 M

Macrophages 73 69 57 51 76 81 79 75

± 6 *# ± 3 *# ± 5# ±3 +_6* ± 4* ± 5* ± 3*

Chemotaxis b

Lymphocytes 78 73 66 47 79 81 79 76

± 6* ± 5* -+ 4 *# ±3 ± 4* ± 3* ± 4* ± 5*

Macrophages 489 554 601 640 1145 858 920 963

± ± ± ± ± ± ± ±

41 *# 39 *# 38 # 38 112" 72"~ 87"~ 81 "9

Lymphocytes 301 403 532 641 451 368 409 473

i ± ± ± ± ± ± ±

49* 71" 46 *~ 46 61" 61"~ 37* 42*

Cells were incubated with 10 10 M PACAP38 or Hank's solution (controls) in the presence or absence of different concentrations of forskolin (FK), and adherence and chemotaxis index were determined as described in Materials and Methods. a: The adherence index was determined after 10 min of incubation, b: The cells were incubated with PACAP38 and/or FK for 3 h in the upper chamber. FMLP (10-8 M) was placed in the lower chamber. Results are the mean + SD of six experiments performed in duplicate. *P < 0.05 (compared to controls), 9p < 0.05 (compared to corresponding PACAP38-treated samples, without forskolin), #P < 0.05 (compared to the corresponding samples treated with PACAP38 plus forskolin)

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tested, between forskolin-treated samples and the corresponding samples incubated with PACAP38 plus forskolin. However, PACAP38 failed to prevent the reduction of the chemotaxis index of peritoneal lymphocytes by forskolin (Table 4). In order to elucidate the specificity of the effects of PACAP38 on adherence and chemotaxis capacity in peritoneal macrophages and lymphocytes and the type of receptor involved in these modulatory effects, we investigated the ability of [N-Ac-Tyr1, D-Phe2]-GRF (1-29) NH2, a VIP-receptor antagonist,26 to modulate the PACAP38 effect on adherence and chemotaxis indices by incubating increasing concentrations of VIP antagonist in the presence of a fixed concentration of PACAP38 (10-1° M). As shown in Table 5, the effects of PACAP38 on adherence and mobility of lymphocytes were dose-dependently inhibited by simultaneous incubation with VIP antagonist. The most effective concentration of antagonist for reduction of the maximal neuropeptide effect was 10-5 M. The lower VIP antagonist concentration used (10-s M) was ineffective in reducing the effect on lymphocyte adherence and chemotaxis by PACAP38. However, the VIP-receptor antagonist had no effect on the stimulation by PACAP38 of the adherence and chemotaxis indices of macrophages, while a significant effect (P< 0.05) was observed in the adherence index of

macrophages treated with the highest antagonist concentration used (10-5 M) (Table 5). The effect of the VIP antagonist on the modulation of adherence and chemotaxis capacity of lymphocytes and macrophages by VIP was used as a positive control for the results obtained with PACAP38, showing similar results in both cases. Interestingly, the VIP antagonist showed a higher effect in the adherence of macrophages treated with VIP than those incubated with PACAP38 (Table 5). Incubation with the VIP antagonist alone at concentrations of 10-s- 10-s M had no effect on the adherence and chemotaxis capacity of peritoneal macrophages and lymphocytes (data not shown). Moreover, the simultaneous incubation with 10-s M PACAP38 and 10-9 VIP did not show statistically significant differences either in lymphocyte and macrophage chemotaxis or in lymphocyte adherence compared with samples treated with PACAP38 alone or VIP alone (Table 5). However, lymphocyte adherence was significantly higher (P< 0.05) in samples treated with PACAP38 plus VIP than in samples treated with PACAP38 or VIP alone (Table 5). DISCUSSION

The effect of PACAP on the immune system is little known and, as far as we know, the data in the present

Table 5 The effect of increasing concentrations of VlP antagonist (N-Ac-Tyr 1, Phe2) - GRF (1-29)-NH~ on the modulation of adherence and chemotaxis of rat peritoneal macrophages and lymphocytes by PACAP38. Adherence a

Chemotaxis b

Treatment Macrophages Controls PACAP38 PACAP38 + PACAP38 + PACAP38 + PACAP38 + VIP VIP + Antag VIP + Antag VIP + Antag VlP + Antag

Antag Antag Antag Antag

104 M 104 M 10-7 M 104 M

10-~ M 104 M 10 -7 M 104 M

51 76 68 72 77 78 70 60 63 66 69

_+3 + 6* + 4*8 + 4* _+5* + 6* _+5* + 4*8 _+4*8 _+5* + 3*

Lymphocytes 47 79 51 61 67 77 67 45 52 56 62

_+3 _+4* + 3*8 _+5*8 + 4*8 _+4* + 5* + 38 + 4 *8 + 4*8 _+4*

Macrophages 640 + 38 1145 _+ 112" 1090 + 101" 1086 -+ 137" 1146 _+102* 1125 + 112* 1090 _+ 123" 1080 _+117" 1072 _+104" 1053 _+127" 1020 _+ 109"

Lymphocytes 641 451 639 597 550 498 411 630 574 525 449

-+ 46 _+61" + 538 -+ 338 _+42*8 _+46* _+37* + 558 + 49*8 _+44*8 + 51"

Peritoneal suspension was incubated with 10-~° M PACAP, 10 -9 M VIP or Hank's solution (controls) in the presence or absence of different concentrations of VIP antagonist (Antag). Adherence index and chemotaxis index were determined as described in Materials and Methods. a: The adherence index was determined after 10 min of incubation, b: The cells were incubated with PACAP38, VIP and/or VIP antagonist for 3 h in the upper chamber. FMLP (104 M) was placed in the lower chamber. Each value is the mean + SD of six experiments performed in duplicate. *P < 0.05 (compared to controls), 8p < 0.05 (compared to corresponding neuropeptide-treated samples, without VIP antagonist). The adherence index of samples treated simultaneously with 104 M PACAP38 and 10-9 M VIP was: 82_+6, in macrophages, and 79+6, in lymphocytes. The chemotaxis index of samples treated simultaneously with 104 M PACAP38 and 104 M VIP was: 1166_+107, in macrophages, and 422_+41, in lymphocytes.

Neuropeptides (1996) 30(6), 583-595

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study are the first on the biological effect of PACAP on adherence and mobility of peritoneal macrophages and lymphocytes studied simultaneously. These are two functions shared by macrophages and lymphocytes27 that represent crucial events in the immune response. This study provides evidence that PACAP38 has a differential action on the mobility of two immune cell types. Thus, PACAP38 caused a stimulatory effect on macrophages and an inhibitory effect on lymphocytes. In previous work, we found that PACAP27, PACAP38 and VIP inhibit, in a similar way, the mobility of murine thymocytes and splenic lymphocytes, 7 while VIP may also show the opposite effect on the mobility of routine peritoneal lymphocytes and macrophages. TM The effects were dose-dependent for a wide range of doses, from 10-~2 to 10-~ M, being observed between 10-~ and 10-7 M with a peak at 10-1° M in macrophages and 10-9 M in lymphocytes. This is a close range at which PACAP has been shown to produce biological effects on thymus and spleen lymphocyte mobility, 7 on thymocyte apoptosis 8 and on other parameters. 3,28 A similar range of concentrations of VIP produces effects on immune cells. 7,1z18This is a related peptide showing a 68% homology with PACAP38P Moreover, PACAP receptors have been found in immune cells. 5,~° Pozo and colleagues (submitted for publication) have recently described the presence of two classes of PACAP binding sites in rat peritoneal macrophages: a class with a high affinity (Kd=0.6 ruM), and a class with low affinity (Kd=0.1 ~M). Thus, the Kd of the high affinity binding sites described for PACAP38 in macrophages (submitted for publication) and lymphocytes 5 corresponds to neuropeptide concentrations (10-9-10 -~° M) that showed the highest regulatory effect in our study. The lower effect caused by the higher concentrations could be due to a process of cell desensitization by internalization of those neuropeptide receptors, 29 which is characteristic of a 'down-regulation' of different receptors. Our results show that PACAP38 produced an enhancement in the adherence capacity of peritoneal macrophages and lymphocytes. Adherence of immune cells to the microvascular endothelium or other tissues represents the initial step in the elicitation of these cells to sites of inflammation or immune response ~4,15and is a property shared by lymphocytes and phagocytic cells. ~ Since adherence of these cells to plastic surfaces seems to resemble closely their spreading to cellular surfaces 2° and represents a significant stimulus for the induction of steady-state levels of several cytokines in monocytes, 3° the increased adherence found here for peritoneal macrophages and lymphocytes could indicate an activation state of immune cells by PACAP38. Using the same technique, other neuropeptides such as VIP/s GRP, ~9 neuropeptide y,31 neurotensin32,33 or cholecystokinin 34 have been shown to increase the activity of those murine © Pearson Professional Ltd 1996

peritoneal cells. However, with the same method, FMLP at 10-8 M decreases the adherence capacity ,of rat peritoneal macrophages and lymphocytes. 18 Although adherence leads to a firm attachment of leukocytes allowing the migration of these cells, le mobility is necessary for carrying out the immune response. In this work, we confirm the effect of PACAP38 as a regulatory peptide that can decrease the traffic of peritoneal lymphocytes, a fact previously observed with murine lymphocytes from thymus and spleen. 7 However, the effect of PACAP38 on macrophages was the opposite: an increased mobility in peritoneal macrophages. Similar results have been obtained with VIE which decreases the :mobility of lymphocytesZ18 and increases that of macrophages. 18,35,36 Other neuropeptides have been identified as chemotaxis stimulators for murine peritoneal macrophages and lymphocytes, such as GRP, neurotensin and neuropeptide y,19,31-33,37 or as chemotaxis inhibitors for both kinds of cells, such as cholecystokinin?4 PACAP38 not only stimulates chemotaxis in macrophages but also acts as a potent chemoattractant itself. This effect occurs at the same concentrations at which PACAP38 induced the other immune functions, suggesting that this chemoattraction may be other physiologically relevant function. However, PACAP38 does not show any effect on the chemoattractant capacity of rat lymphocytes from peritoneum. In previous work, PACAP38 was also shown to be ineffective as a chemoattractant for thymus and spleen lymphocytes. 7 Other neuropeptides, such as VIP, 18 substance p38 or bombesinqike peptides, 37 have been shown to be chemoattractants for phagocytic cells. In contrast, neurotensin and neuromedin N stimulate chemotaxis of peritoneal lymphocytes and macrophages, but they do not behave as chemoattractants for them. 32,33 The results found with PACAP38 in the present work are very similar to those obtained by us with VIP. 18VIP, at the same concentration range as PACAP38, stimulated the adherence of peritoneal macrophages and lymphocytes, increasing macrophage mobility but decreasing this activity in lymphocytes. Interestingly, the maximum effect of VIP was produced at 10-9 M, one range of concentration higher than PACAP38, in both peritoneal cells, with a slightly smaller effect. This is in accordance with the Kd and diacylglycerol production observed for VIP in peritoneal macrophages (submitted for publication). The explanation of the opposite effect of PACAP38 on lymphocyte and macrophage mobility should be found in the particular receptor and intracellular mechanisms that PACAP use in each type of cell. Two PACAP receptor types have been described, the selective receptor type I, which prefers PACAP38 and PACAP27 over VIE and type II (also named common VIP/PACAP receptor) which has an approximately equal high affinity for PACAP38, Neuropeptides (1996) 30(6), 583-595

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PACAP27 and VIP, and which is considered as the classical VIP receptor.3,9,1°,12,28 Likewise VIP receptors, types I and II PACAP receptors, are members of the secretin family of receptors linked to G proteins of the Gs family and thus activate adenylate cyclase. Moreover, type I PACAP receptor may also interact with a protein of the Gq family linked to the activation of phospholipase C (PLC).13 In this sense, recent experiments performed in our laboratory have shown gene expression of the type I PACAP receptor in rat peritoneal macrophages. This receptor seems to be coupled to the inositol phospholipid pathway, because PACAP38, in a concentration range from 10-1~ to 10-~ M, caused a stimulation of 1,2-diacylglycerol production in peritoneal macrophages with an EDso about nM (submitted for publication). Thus, peritoneal macrophages have type I and type II PACAP receptors whereas peritoneal lymphocytes only have the type II PACAP receptor. With respect to the intracellular mechanisms that PACAP uses in immune cells, the implication of cAMP as a second messenger seems evident, since this neuropeptide is a potent stimulator of cAMP production in anterior pituitary cells 2 and we found that PACAP increases cAMP levels in thymus and spleen lymphocytes. 7 It is well known that agents which increase the intracellular cAMP levels inhibit the functions of a variety of effector cells involved in the immune reaction. 39 Thus, cAMP or an increase of cAMP has been reported to decrease phagocytic functions 4°-42 and is an inhibitor of lymphocyte and phagocyte chemotaxis, 43-4~as well as of lymphocytic proliferation,4z4~ and IL-2 production. 49,5° Thus, neuropeptides that increase cAMP levels decrease immune functions, ~8,34 and inversely, agents and neuropeptides that decrease cAMP levels cause an increase in immune function. ~9,3~,4~VIP, the neuropeptide related to PACAP, uses cAMP as a second messenger in lymphocytes.Zl~,5~ In addition, biochemical and pharmacological studies have shown the presence of VIP receptors in lymphocytes 52,53 and in macrophages. 54,55 Recently, we have described VIP receptor expression in rat peritoneal macrophages and lymphocytes by RT-PCR analysis? 6 These VIP receptors are linked to activation of adenylate cyclase and elevation of intracellular cAMP levels? ~.szFor this reason, the inhibitory action of VIP on the different immune functions has been attributed to its capacity to increase cAMP levels? 8 For the same reason, PACAP could also inhibit immune functions. Thus, the inhibition of mitogen-stimulated proliferation of spleen lymphocytes and of mobility of lymphocytes from thymus and spleen by PACAP have been reported. 5,z In this study, the incubation of macrophages or lymphocytes with forskolin, an enhancer of cAMP levels,24 decreased the chemotaxis in these cells. Similar results were obtained by us in previous work. z~a However, the Neuropeptides (1996) 30(6), 583-595

presence of PACAP38 in samples incubated with forskolin increased the chemotaxis in macrophages in relation to the values obtained with only forskolin or those obtained on controls. In lymphocytes the presence of PACAP38 did not counteract the effect of forskolin, suggesting a different pathway for PACAP38 in lymphocytes and macrophages. In fact, VIP, which uses cAMP as a second messenger in lymphocytes, acts through PKC activation in rat peritoneal macrophages. 17 It is well known that agents that act through production of diacylglycerol and inositol triphosphate by PLC, with subsequent PKC activation and increase of intracellular calcium levels, stimulate immune cell functions? 9,6° Thus, immune cell mobility,~,62 and activation by chemoattractants such as formyl-Met-Leu-Phe peptide 63 are mediated by PKC activation. Several neuropeptides that enhance inositol triphosphate synthesis, PKC activation or intracellular calcium concentration, such as GRP, neuromedin N or neuropepfide y,19,3~2 stimulate phagocytic cell functions like chemotaxis. Neuropeptides that decrease PKC activity, such as CCK, 34inhibit these functions. In the present study, incubation with increasing concentrations of PMA, a specific PKC activator, showed a progressive enhancement of the chemotaxis of lymphocytes and macrophages, whereas retinal, an inhibitor of PKC, 25 decreased it. The simultaneous presence of PMA and PACAP38 was not synergistic for the stimulatory effect of PMA on macrophage chemotaxis, whereas the increase of this function in lymphocytes by PMA was partially prevented. However, PACAP38- retinal combinations resulted in a partial suppression of the retinal-inhibition of chemotaxis in macrophages and a synergistic effect in lymphocytes. Therefore, since PACAP38 decreased chemotaxis in peritoneal lymphocytes and increased it in macrophages, we think that PACAP38 could use two different physiological receptors, linked principally to adenylate cyclase in lymphocytes and to phospholipase C in macrophages. Thus, in macrophages is possible that PACAP38 acts through the type I PACAP receptor linked to activation of phospholipase C, 13 and in lymphocytes through the type II PACAP receptor, the only type of PACAP receptor in lymphocytes. The reason for the increase of adherence capacity by PACAP38 in macrophages and lymphocytes is that this activity can be mediated by PKC and by cAMP-dependent kinase (PKA)?4 In this study, PMA produced an augmentation and retinal produced a reduction in both types of cell. The effect of PMA was higher in the adherence index of macrophages than in that of lymphocytes. Moreover, incubation of macrophages and lymphocytes with forskolin resulted in an increase in the adherence capacity, which was higher in lymphocytes than in macrophages. Thus, the stimulation of adherence in © Pearson Professional Ltd 1996

PACAP modulates adherence and mobility 593

macrophages by PACAP38 could implicate PKC activation whereas in lymphocytes it could implicate PICA activity. This is confirmed with data obtained from simultaneous incubations with PACAP38 and PMA, PACAP38 and retinal, and PACAP38 and forskolin. The effects of PACAP38 and PMA were not additive in either macrophages or lymphocytes and the partial loss of the inhibitory effect of retinal by PACAP38 in macrophages was smaller than that in lymphocytes. Moreover, the simultaneous presence of PACAP38 and forskolin was somewhat synergistic on both peritoneal cell types, although in lyrnphocytes the potentiation was smaller than in macrophages. In order to demonstrate the specificity of the effects observed and to provide additional evidence for the receptor-mediated nature of the response produced by PACAP38, we have also investigated the ability of the [N-Ac-Tyr 1, D-Phe2]-GRF (1-29) NH v a VIP receptor antagonist, 26 to modulate the effect of PACAP38 on lymphocyte and macrophage chemotaxis. This GRF analog is a VIP receptor antagonist that competitively antagonizes VIP/PACAP actions and selectively inhibits neuropeptidestimulated adenylate cyclase?6,6s,66 The stimulatory action of PACAP38 on lymphocyte adherence was prevented by all VIP antagonist concentrations tested while macrophage adherence was only partially prevented by the highest antagonist concentration used (10-5 M). There was a slight synergistic effect of VIP and PACAP in macrophage but not in lymphocyte adherence. Thus, since VIP/PACAP receptor (type II PACAP receptor) was present in peritoneal macrophagesy it is probable that PACAP38 exerts its action on adherence through binding to type II PACAP receptors and[ elevation of intracellular cAMP levels in lymphocytes, while in macrophages PACAP38 acts mainly through binding to type I PACAP receptor and PKC activation, and partly through binding to type II PACAP receptor and PICA activation. On the other hand, the present study showed the ability of this VIP antagonist to partially reverse the inhibitory effect of PACAP38 on the chemotaxis of peritoneal lymphocytes, but not of macrophages. The effect of this antagonist was dose-dependent, in a range similar to that reported previously in thymocyte and splenocyte chemotaxis/ in thymocyte glucocorticoid-induced apoptosis, s in IL-10 production 67 and in other processes. 65,66Since the VIP antagonist used antagonizes neuropeptide actions at the receptor level, we suggest that PACAP38 exerts its action on peritoneal lymphocyte chemotaxis through the common VIP/PACAP receptor (type II PACAP receptor). Thus, the opposite effect of PACAP38 on chemotaxis of peritoneal macrophages and lymphocytes could be explained by the action of PACAP38 on macrophages, through PKC activation by binding to type I PACAP receptor and on lymphocyte,s, through adenylate cylase activation by binding to VIP or type II PACAP receptor. © Pearson Professional Ltd 1996

From a physiological viewpoint the fact tlhat PACAP can produce a different action on peritoneal lymphocytes and macrophages, two types of immune cells that share the same localization, could be of great interest in the understanding of the complex regulation network that underlies the modulation of immune response by the nervous system. ACKNOWLEDGEMENTS

We thank Dr J. Miquel for correcting this manuscript. This work was supported by DGICYT, Grant No. PB94-0310, from the Ministerio de Education y Ciencia, and by FISss, Grant No. 96/1059, from the Ministerio de Sanidad y Seguridad Social, Spain.

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