Calcium Signaling in Pancreatic Immune Cells In situ

Abstract Immune cells were identified in intact live mouse pancreatic lobules and their Ca2+ signals, evoked by various agents, characterized and compared with the simultaneously recorded Ca2+ signals in neighboring acinar and stellate cells. Immunochemistry in the live lobules indicated that the pancreatic immune cells most likely are macrophages. In the normal pancreas the density of these cells is very low, but induction of acute pancreatitis (AP), by a combination of ethanol and fatty acids, markedly increased the number of the immune cells. The principal agent eliciting Ca2+ signals in the pancreatic immune cells was ATP, but these cells also frequently produced Ca2+ signals in response to acetylcholine and to high concentrations of bradykinin. Pharmacological studies, using specific purinergic agonists and antagonists, indicated that the ATP-elicited Ca2+ signals were mediated by both P2Y1 and P2Y13 receptors. The pancreatic immune cells were not electrically excitable and the Ca2+ signals generated by ATP were primarily due to release of Ca2+ from internal stores followed by store-operated Ca2+ entry through Ca2+ release-activated Ca2+ channels. The ATP-induced intracellular Ca2+ liberation was dependent on both IP3 generation and IP3 receptors. We propose that the ATP-elicited Ca2+ signal generation in the pancreatic immune cells is likely to play an important role in the severe inflammatory response to the primary injury of the acinar cells that occurs in AP.


Introduction
Cytosolic Ca 2þ signals in the acinar cells of the exocrine pancreas, evoked by acetylcholine (ACh) or cholecystokinin (CCK), control the physiologically important secretion of digestive enzymes and fluid. 1 The mechanisms responsible for the primary intracellular Ca 2þ release and the subsequent Ca 2þ release-activated Ca 2þ (CRAC) entry of Ca 2þ are well established. [1][2][3][4] Although the vast majority of Ca 2þ signaling studies have been conducted on acutely isolated mouse acinar cells or small acinar cell clusters, the general validity of the results obtained has been confirmed by studies in more intact preparations as well as in experiments on human acinar cells. 1,5 Whereas local repetitive Ca 2þ rises regulate physiological secretion, global and sustained elevations of the cytosolic Ca 2þ concentration ([Ca 2þ ] i ) initiate the disease acute pancreatitis (AP). 6 Such excessive Ca 2þ signals can be elicited by a combination of ethanol and long-chain fatty acids, bile acids, or be drug-induced, for example by Asparaginase. 7 The sustained global elevation of [Ca 2þ ] i is generally maintained by open CRAC channels, 2,6 but can also occur via pressure-induced Piezo1 activation of TRPV4 channels. 8 Given that the acinar cells constitute the bulk of the exocrine pancreatic tissue and that these cells synthesize and secrete the digestive enzymes in response to food intake and are responsible for the initiation of AP, it is not surprising that Ca 2þ signaling studies in the exocrine pancreas have largely been confined to these cells. However, it has recently become clear that other cell types in the exocrine pancreas also play a role, particularly in the pathophysiology of AP. 4,[9][10][11][12] Employing isolated lobules of the exocrine pancreas, in which the normal microscopic structure of the acinar environment is preserved, it has been possible to record simultaneously cytosolic Ca 2þ signals in several different cell types, including stellate cells and intrinsic nerves. 10,11 Whereas the role of the Ca 2þ signals evoked by ACh and CCK in the acinar cells is well understood, the physiological importance of the Ca 2þ signals evoked by bradykinin (BK) in the stellate cells 10 is unclear. There is, however, evidence indicating that BK-elicited Ca 2þ signals in the stellate cells can magnify the damage to the acinar cells caused by various agents inducing AP, including the combination of alcohol and fatty acids as well as bile acids. [9][10][11] At least part of the damaging effect of the BK-elicited Ca 2þ signals in the stellate cells would appear to be mediated by NO generation, due to Ca 2þ -activation of NO synthase in these cells. 12 In our recent work on Ca 2þ signaling in the peri-acinar environment, 11 we identified, in addition to stellate cells and nerve cells, an unknown cell type, which we called X-cells. These cells displayed prominent Ca 2þ signals in response to stimulation with ATP and have been the focus of the present study. We now show evidence indicating that these X-cells are pancreatic immune cells, most likely macrophages.
Previously, macrophages of the exocrine pancreas have been studied in culture or fixed tissue, primarily under pathological conditions such as ductal ligation, carbon tetrachloride-induced pancreatitis, and experimental pancreatic cancer. 13 During pancreatic injury, macrophages infiltrate the tissue leading to inflammation, tissue destruction, and high rates of morbidity and mortality. 14 There have been limited studies in normal pancreatic tissue that possesses different types of tissue-resident myeloid cells. They could be identified by immunohistochemistry using specific surface proteins including CD11b and F4/80 that are highly expressed specifically in macrophages. [15][16][17] It was, however, difficult to identify more precisely myeloid cell subpopulations in the exocrine tissue due to cross-reactivity of conventional dendritic cells and macrophages to the same tissue antigens. 17 Here we now characterize the Ca 2þ signaling properties of macrophages in situ, using freshly isolated lobules of exocrine pancreas. We demonstrate vigorous Ca 2þ signal generation induced by ATP as well as some other agents. As ATP will be released from damaged acinar cells in the very early stage of AP, such Ca 2þ signals could play an important role in the generation of the inflammatory response, which is the major cause of the destruction of the pancreas and surrounding tissues in AP. 18

Ethical Approval
All animal studies were ethically reviewed and conducted according to the UK Animals (Scientific Procedures) Act, 1986. All animal procedures and experimental protocols were performed under a Project Licence granted by the UK Home Office and approved by the Animal Care and Ethics Committees at Cardiff School of Biosciences, Cardiff University. Animals were maintained in plastic cages supplied with fresh corn cob bedding, tap water, and commercial pelleted diet.

Induction and Evaluation of Experimental AP
The alcohol-induced experimental model of AP was induced in C57BL6/J mice that received two intraperitoneal (IP) injections of PBS followed by IP injections of a mixture of ethanol (1.35 g/kg) and palmitoleic acid (150 mg/kg) at 1 h intervals as previously described. 11 We refer to this AP model as FAEE-AP, since fatty acids and ethanol can react together inside cells to produce fatty acid ethyl esters (FAEEs). Control mice received IP injections of the PBS solution alone. Mice were humanely killed by cervical dislocation (Schedule 1) 48 or 72 h after the last injection. For histological assessment pancreatic tissue was fixed in 4% formaldehyde and embedded in paraffin. Fixed slices (4 lm thickness) stained with hematoxylin and eosin, !10 random fields of view (magnification: Â200), were evaluated by two blinded independent investigators grading (scale, 0-3; means 6 SEM; n ¼ 3 mice per group) edema, inflammatory cell infiltration, and necrosis as previously described. 19

Lobule Preparation
Pancreatic lobules were freshly isolated from the pancreas of 5to 7-week-old male C57BL6/J mice 10 or from mice in which FAEE-AP had been induced as described above. The pancreas was rapidly removed, injected with standard Na þ -Hepes-based solution containing collagenase and incubated for 5-6 min at 37 C. The standard solution was composed of (in mM): NaCl, 140; KCl, 4.8; Hepes, 10; MgCl 2 , 1; glucose, 10; CaCl 2 , 1 (unless stated otherwise), pH 7.3 (NaOH). The standard solution in experiments for investigation of the effects of membrane depolarisation was modified to contain 100 mM KCl and 44.8 mM NaCl. All experiments were carried out with pancreatic lobules attached to the coverslip of a perfusion chamber at room temperature ($23 C).

Ca 21 Measurements
Pancreatic lobules were loaded with 5 lM Fluo-4 acetoxymethyl (AM) ester for 20 min at room temperature. The lobules were transferred into a flow chamber and perfused with the standard solution alone or containing different chemicals as described in the experimental protocols of the result section. Cells were visualized using a Leica SP5 MPII two-photon confocal microscope, with an x63 1.2NA objective lens. The Fluo-4 excitation wavelength was 488 nm and emission was collected at 500-560 nm with resolution of 256x256 pixels and speed of 0.7 frames/s. Images were analyzed using Leica Confocal Software (Leica, Mannheim, Germany). Fluorescence signals were plotted as normalized F/F 0 . Control Immunoglobulin G (IgG) was used in concentrations of 0.1-0.25 mg/mL. ANOVA or Student's t-test was performed for statistical analysis.

Immunostaining in Ex vivo Lobules
Immunostaining of live pancreatic lobules was performed as previously described. 10 Mouse F4/80 and mouse CD11b/Integrin alpha M Alexa Fluor V R 647-conjugated monoclonal rat antibodies were used to label specific surface proteins of immune cells, usually at the end of Ca 2þ measurement experiments, unless otherwise stated. After blocking with 1% BSA and 10% goat serum containing PBS, the isolated pancreatic lobules were incubated for 1 h at room temperature with the selected antibody. Antibody staining was visualized by exciting Alexa Fluor V R 647 with 633 nm laser at 10% power and emitted light was collected at 640-700 nm. Hoechst 33342 was used to determine the position of nuclei using excitation 405 nm and collecting light at 420-480 nm. Conjugated antibody fluorescence was also overlaid with Fluo-4 staining as described in the Ca 2þ measurements section. Lobules were attached to the glass coverslips covered with poly-L-lysin.

X-cells Identified as Pancreatic Immune Cells
In our previous study, 11 the unidentified X-cells displayed Ca 2þ signals that were distinct from those observed in the wellknown cells of the pancreatic lobules. One possibility, which we have now investigated, is that the X-cells might be immune cells, as such cells have occasionally been found in pancreatic tissue. 13,17 To test the hypothesis that the X-cells previously described 11 are immune cells, we employed immunostaining with different antibodies against surface proteins of immune cells labeled with the fluorescent indicator Alexa Fluor 647 (Figures 1A-C and 2D) to allow post staining at the end of functional experiments (Figure1A(iii) and Figure1C(iii)). The fluorescently labeled antibodies F4/80 as well as CD11b have been commonly used to label macrophages. 13,20 Staining confirmed the identity of X-cells as pancreatic macrophages (PMs) in the pancreas (Figures 1A-C and 2E). In addition, the ear-like shape of nuclei, typical for macrophages, monocytes, eosinophils, and dendritic cells [21][22][23][24] was very different from the classical round shape of nuclei observed in other cells of the pancreas when stained with Hoechst 33342 ( Figure 1A, B).
The control antibodies IgG are known to induce Ca 2þ spikes in activated immune cells. 25 We applied IgG to control pancreatic lobules, but did not detect any oscillations. Instead we observed occasionally single Ca 2þ spikes ( Figure 2A), but their appearance was independent of the presence of IgG. We then tested the effect of IgG on PMs in our AP model (FAEE-AP). In lobules isolated from FAEE-AP pancreas (48 h), we detected IgG-induced Ca 2þ oscillations, after a substantial delay, in about 30% of PMs ( Figure 2B). Without IgG stimulation, no oscillations were observed in PMs from FAEE-AP pancreas. These data indicate that PMs are largely quiescent in the pancreatic tissue of control mice, only displaying the occasional spontaneous Ca 2þ spike. However, after AP induction, PMs become activated 20 and now close to one-third of the cells respond to IgG stimulation with repetitive Ca 2þ spiking (oscillations) ( Figure 2B, C). The duration of the spikes in the FAAE-AP pancreas appeared to be slightly longer than in the control situation, but the difference was not significant ( Figure 2D). The amplitudes of the Ca 2þ spikes in the FAEE-AP pancreas were not significantly different from those observed in the control pancreas (P > 0.06).
Immunostaining of PMs with the fluorescently labeled antibodies F4/80 against surface IC proteins ( Figure 1A-C) was used to calculate the relative density of PMs ( Figure 2E, F). Whereas the density of pancreatic stellate cells (PSCs) was not different in tissues isolated from control and FAEE-AP (72 h) mice (n ¼ 14 and n ¼ 31, P > 0.54), the density of PMs increased significantly in FAAE-AP mice after 48 h (P < 0.033) and was highly significant after 72 h (P < 0.0001; Figure 2E, F).

ATP-elicited Ca 21 Signals in PMs
We have previously reported that X-cells, now identified as PMs, are highly responsive to stimulation with micromolar concentrations of ATP. 11 Figure 3A-D demonstrate that PMs (green traces) are capable of generating substantial Ca 2þ signals in response to ATP (10 lM) ( Figure 3A-D).
We tested the possibility that the PMs might be electrically excitable. In these experiments, the cells in the lobule were depolarized by exposure to a solution with a high (100 mM) K þ concentration (HK þ ) as previously described. 11 This evoked short-lasting Ca 2þ signals in about 30% of the PMs ( Figure 3A), but this could be an indirect effect due to release of ACh from depolarized intrinsic nerves (dark blue trace in Figure 3A). In the presence of atropine (1mM) HK þ failed to evoke any elevation of [Ca 2þ ] i in the PMs, whereas the Ca 2þ signals evoked by ATP and BK were preserved ( Figure 3C). ACh induced Ca 2þ signals in a majority of PMs ($70%) ( Figure 3B), whereas CCK ( Figure 3A) had no effect. Bradykinin elicited Ca 2þ signals in PMs (in $40% of the cases), which were delayed compared to the responses in the neighboring PSCs ( Figure 3C).
Depletion of intracellular stores by prolonged stimulation with 10 lM ATP, in the absence of external Ca 2þ (and in the presence of ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0tetraacetic acid (EGTA) (0.5 mM)), lead to the cessation of Ca 2þ signaling ( Figure 3D). Ca 2þ oscillations resumed after reintroduction of 1 mM Ca 2þ to the bath solution and removal of EGTA ( Figure 3D).
To investigate the potential involvement of inositol trisphosphate (IP 3 ) receptors in ATP-elicited Ca 2þ signaling in PMs, we treated pancreatic lobules with either 100 lM 2-APB ( Figure 4A) or the phospholipase C inhibitor U 73122 (10 lM) ( Figure 4B). In both cases this resulted in almost complete inhibition of ATP-elicited Ca 2þ signal generation (Figure 4). In contrast, application of an inhibitor of adenylyl cyclase, SQ 22536 (200 lM), only had a partial but nevertheless significant inhibitory effect on Ca 2þ signals evoked by 1 lM ATP, but had no effect on the responses to higher doses of ATP, i.e. 3 and 10 mM ( Figure 4C).
As mentioned above ( Figure 3D), the ATP-elicited Ca 2þ signals in PMs depend predominantly on Ca 2þ release from internal stores that can be depleted by prolonged stimulation with ATP in the absence of external Ca 2þ . To study the replenishment of  internal stores we have used a standard cyclopiazonic acid (CPA) protocol for Ca 2þ store depletion, while observing simultaneously four different cells (two PMs, pancreatic stellate cells (PSCs), and Pancreatic acinar cells (PACs); Figure 5A). Application of ATP at the beginning of the experiment, before CPA addition, induced typical Ca 2þ signals in both PMs, a smaller response in the PACs and no response in the PSCs ( Figure 5A). After addition of CPA and removal of external Ca 2þ , there was no response to ATP in the PAC and the ATP-elicited Ca 2þ signals in the PMs were gradually reduced and finally almost disappeared ( Figure 5A). Readmission of external Ca 2þ (1 mM) induced rapid Ca 2þ elevations in all cells ( Figure 5A) most likely due to store-operated Ca 2þ entry. In a similar type of experiment ( Figure 5B), elevations of [Ca 2þ ] i in three different cells (PMs, PACs, and PSCs) were observed after both short and continuous external readmissions of 1 mM CaCl 2 . After readmission of external Ca 2þ there was also recovery of the ATP-induced response in the PM ( Figure 5B). The most likely explanation for the observed rise in [Ca 2þ ] i immediately upon external Ca 2þ readmission is that CRAC channels are open as a result of the intracellular Ca 2þ store depletion following blockade of the SERCA pumps by CPA. 2 Antigen stimulation of immune cells is known to trigger Ca 2þ entry through CRAC channels, promoting the immune response to pathogens. 26 GSK 7975A (10 lM), a well-known CRAC channel blocker, 2 abolished Ca 2þ readmission-induced Ca 2þ entry in the PM ( Figure 5C, green trace), whereas there was an incomplete, but substantial, inhibition of Ca 2þ entry in the PSC ( Figure 5C, red trace), in agreement with our previous findings. 11

Pharmacology of ATP-elicited Ca 21 Signaling in PMs
The purinergic agonist MeSADP, which mainly acts on P2Y1, P2Y12, and P2Y13 receptors, 27 elicited Ca 2þ signals in PMs ( Figure 6A-D). MRS 2179, a selective antagonist of P2Y1  receptors, completely blocked Ca 2þ transients in PMs induced by either ATP or MeSADP ( Figure 6C). However, MRS 2211, a competitive antagonist of P2Y13 receptors also blocked both ATP and MeSADP responses in PMs ( Figure 6D). A selective P2Y1 agonist, MRS 2365, as well as ADP, induced Ca 2þ signals in PMs ( Figure 6B), whereas suramin, a potent blocker of P2Y purinergic receptors, blocked Ca 2þ signals in response to application of 10 lM ATP in PMs (n ¼ 3). We suggest that this may be due to co-operation between P2Y1 and P2Y13 in PMs, as previously reported for mesenchymal stromal cells in adipose tissue. 27

BK-elicited Ca 21 Signals in PMs
We have previously reported that a low concentration of BK (1 nM), which typically elicits clear Ca 2þ signals in PSCs did not induce any changes in the cytosolic Ca 2þ concentration ([Ca 2þ ] i ) in X-cells (PMs). 11 However, in the present study, we found that in about 40% of PMs tested, there was a Ca 2þ signal in response to higher concentrations of BK ( Figure 7A, B). Figure 7B shows typical Ca 2þ signals in PMs elicited by BK at concentrations from 3 nM to 30 nM, but not at 1 nM BK. The BK-induced Ca 2þ signals in PMs were delayed as compared to those in PSCs ( Figure 7A, C). The delay depended on the BK concentration and became progressively shorter at higher concentrations ( Figure 7B). The B2 receptor antagonist WIN 64338 reversibly inhibited the responses to BK ( Figure 7C) in both PMs and PSCs, suggesting that, similar to our previous finding for PCSs, 11 PMs possess the type 2 BK (B2) receptor. Atropine ( Figure 3C) had no effect on the BK-induced responses in PMs.

Discussion
In the quest to understand the mechanisms governing the function of an organ, it is natural initially to focus on the dominant cell type, particularly if it is clear that it is the executor of the organ's primary physiological task. In the exocrine pancreas, it has been clear for a long time that the quantitatively dominating acinar cells synthesize the whole range of enzymes required for the digestion of food entering the gut and that these cells secrete these enzymes in a manner precisely controlled by local repetitive cytosolic Ca 2þ spikes. 1 It is also clear that AP is initiated in the acinar cells by excessive global and sustained elevations of [Ca 2þ ] i . 6 However, in recent years it has become clear that there are other cell types in the peri-acinar environment that can be regarded as "background actors" and there is increasing interest in understanding their function and how they may interact with the principal cells. 28 Given that cytosolic Ca 2þ signal generation is one of the most fundamental mechanisms regulating physiological processes [29][30][31] and that such signals can now be readily recorded in the quasi-intact pancreas, 10,11 Ca 2þ measurements in the peri-acinar cells could be an important tool for obtaining information about the signaling functions of these accessory and less common cell types.
Having recently characterized the Ca 2þ signaling properties of the pancreatic stellate cells (PSCs) in pancreatic lobules and assessed their potential influence on the acinar cells, 10,11 we have now turned our attention to the immune cells in the exocrine pancreatic tissue. We show that these cells exist in low density in the normal pancreas and confirm that their number increases markedly in the first few days following induction of AP by a combination of ethanol and fatty acids ( Figure 2).
Our data for the first time demonstrate functional responses of immune cells in their natural environment, namely in live pancreatic tissue. Ca 2þ measurements in ex vivo lobules have been complemented by immunocytochemistry to test the identity of the monitored cells. 10,11 This technique has allowed us to study both physiological and pathological responses related to AP. Live immunocytochemistry is ideal for primary antibodies labeled with a fluorescent marker, in our case Alexa 647, but could also be carried out with the use of secondary antibodies as previously reported. 10,11 The positive staining with antibodies against F4/80 and CD11b (Figures 1 and 2) indicates that these immune cells are most likely PMs, 32 which have previously been reported to increase in numbers in chronic pancreatitis in mice. 20 Macrophages are known to express a variety of purinergic P2Y receptors 33 and here we show that both ADP and ATP activate P2Y receptors blocked by suramin and specific inhibitors of either P2Y1 or P2Y13 receptors ( Figure 6). The effects of ATP and MeSADP (agonist of P2Y1, P2Y12, and P2Y13 receptors) were abolished by either MRS2179, a selective antagonist of P2Y1 receptors, or MRS 2211, a competitive antagonist of P2Y13. 27 Similar synergistic responses have previously been described for ADP in mesenchymal stromal cells, 27 which required both P2Y1 and P2Y13 receptors, while specific inhibition of either of them abolished the responses to ADP.
It has been known for a long time that macrophages can generate cytosolic Ca 2þ signals via activation of IP 3 receptors 34 and here we show that the ATP-induced Ca 2þ signals in PMs are  primarily due to intracellular Ca 2þ release ( Figure 3) and can be very markedly reduced by the phospholipase C inhibitor U73122 or the inhibitor of store-operated Ca 2þ entry 2-APB (Figure 4), which can also inhibit IP 3 -elicited Ca 2þ release. 35 It is well established that macrophages produce and secrete a wide range of inflammatory cytokines and Ca 2þ signals in these cells have been linked to both production and secretion of these inflammatory mediators. 33,[36][37][38] Although we currently have no data about the consequence(s) of Ca 2þ signal generation specifically in the macrophages in the intact pancreas, it is likely-based on what is generally known about macrophage function, 33,36-38 that the Ca 2þ signals described in our study could play an import role in the generation of the cytokine storm that is such an important feature of severe AP. 39 It has very recently been noted that there are interesting similarities between the multi-organ failures and patterns of elevated cytokines observed in severe cases of COVID-19 and AP. 39 Furthermore, it has been suggested that in addition to the cytokine storm, there is also a BK storm involved in severe cases of COVID-19. 40 This may also be the case in severe cases of AP. Plasma levels of BK are elevated in AP, 41 and this causes Ca 2þ signal-mediated NO formation in the PSCs. [10][11][12] There is evidence indicating that the NO formation amplifies acinar necrosis. 11,12 In macrophages, intracellular Ca 2þ can also activate NO production. 36 It is therefore possible that ATP-elicited Ca 2þ signals in the PMs, via NO formation, could participate in the vicious circle previously described in relation to the interaction between acinar and stellate cells. 11 The cytokine storm is likely to be amplified by the BK storm, since we have shown in this study that the PMs are not only activated by ATP, but also by BK.
Initial damage of the acinar cells, elicited by combinations of fatty acids and ethanol, by bile acids or by asparaginase 6,7,11 would release ATP into the acinar environment and elicit Ca 2þ signals in the pancreatic macrophages. Via NO formation (and possibly also other mechanisms), further damage of the pancreatic acinar cells would occur with additional release of ATP establishing a vicious circle. An initial element of inflammation would induce entry of more PMs into the pancreatic tissue, amplifying the overall impact of Ca 2þ signal generation in these cells and further worsening the severity of AP.
Clearly, we are still at an early stage of understanding the initiation of the inflammatory response that in severe cases of AP results in destruction of the pancreas and other organs, but we can now begin to appreciate the importance of the role that cells in the peri-acinar environment, such as stellate cells and PMs, could play in this process.