Circulating immune complexes of calves with ...

Research in Veterinary Science 106 (2016) 135–142

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Circulating immune complexes of calves with bronchopneumonia modulate the function of peripheral blood leukocytes: In vitro evaluation Marijana Buač a, Slavko Mojsilović a, Dušan Mišić b, Dejan Vuković c, Olivera Savić d, Olivera Valčić b, Dragana Marković a, Dragan Gvozdić b, Vesna Ilić a,1, Natalija Fratrić b,⁎,1 a

Institute for Medical Research, University of Belgrade, Dr Subotića 4 POB 39, 11129, Belgrade 102, Serbia Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobodjenja 18, 11000 Belgrade, Serbia PKB Corporation, Industrijsko naselje BB, 11213 Padinska Skela, Belgrade, Serbia d Institute for Blood Transfusion of Serbia, Svetog Save 39, 11000 Belgrade, Serbia b c

a r t i c l e

i n f o

Article history: Received 8 September 2015 Received in revised form 9 March 2016 Accepted 3 April 2016 Available online xxxx Keywords: Calf bronchopneumonia Circulating immune complexes Peripheral blood leukocytes

a b s t r a c t In this work we studied if circulating immune complexes (CIC) of calves with bronchopneumonia have the capacity to modulate function of peripheral blood leukocytes of healthy cattle. CIC of three month old calves (6 healthy and 6 diseased) were isolated by PEG precipitation. Peripheral blood mononuclear cells (MNCs) and granulocytes from healthy calves and cows were the CIC responder cells in in vitro tests. The most remarkable increase of adhesiveness to polystyrene and ROS synthesis (assessed by NBT test) was detected in cows' granulocytes stimulated with CIC of diseased calves. Results of MTT test showed that CIC of both healthy and diseased calves reduced granulocytes' viability. The strongest effect of inhibition of cows' granulocytes resulted from CIC of diseased calves. CIC only moderately reduced spontaneous viability of calves' MNCs. Again, the strongest effect of CIC isolated from diseased calves was observed. In contrast to the low impact of CIC on non-stimulated cells, their inhibitory effect on viability of mitogen stimulated MNCs was very strong. With CFSE assay we showed that both types of CIC stimulated spontaneous, but inhibited mitogen induced proliferation of calves' MNCs. Propidium iodide staining reviled that CIC increased apoptosis/necrosis of both non-stimulated and mitogen stimulated MNCs. CIC of both healthy and diseased calves modulated the function of peripheral blood MNCs and granulocytes, but a stronger effect of CIC of diseased calves was shown. The age of the donors (calves or cows) of the responder cells, and the activation state of these cells, were also of influence. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Calf bronchopneumonia is a complex disease caused by viruses (BHV-1, PI-3V, BVDV, BRSV) or/and bacteria (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus, Mycoplasma bovis). These infectious agents are ubiquitous in cattle populations, and the disease occurs when the calves' immune response is diminished by environmental stress factors (Mosier, 2014). The important role of humoral immunity in the defense was confirmed by data showing that the highest incidence of pneumonia occurs in calves aged two to four months. At this age the concentration of total serum immunoglobulins (Corbeil et al., 1984), and IgG specific for some of the pathogens associated with bronchopneumonia (Prado et al., 2006) are at their lowest level, due to an almost complete degradation of passively acquired maternal IgG and insufficient synthesis of their own antibodies. Numerous ⁎ Corresponding author. E-mail address: [email protected] (N. Fratrić). 1 Joint principal investigators.

http://dx.doi.org/10.1016/j.rvsc.2016.04.002 0034-5288/© 2016 Elsevier Ltd. All rights reserved.

studies confirmed, directly or indirectly, that complexes of causative antigens and specific antibodies (i.e. immune complexes) are of importance for the pathogenesis of bronchopneumonia in humans (Mizutani and Mizutani, 1986; Mellencamp et al., 1987; Monsalvo et al., 2011). Deposits of immune-complexes in the lung tissue, together with histological finding of massive inflammation (McBride et al., 1999; Mulongo et al., 2015) indicate that immune-complexes play a role in the pathogenesis of calf bronchopneumonia. Studies conducted on rodent models (reviewed in Gao et al., 2006; Ward, 2010) showed that intra-alveolar deposition of IgG immune complexes activate complement system and lung resident macrophages and, recruit and activate blood neutrophils. The activated cells secreted pro-inflammatory cytokines, reactive oxygen species (ROS) and proteinases which trigger acute lung injury. The immune-complexes mediated lung inflammation is normally terminated endogenously by anti-inflammatory cytokines, and inhibitors of leukocyte proteinase and matrix metalloproteases which mediate the repair of injured lung tissue. However, an imprecise regulation of pro-inflammatory and anti-inflammatory components in the injured lung tissue are critical for the pathogenesis of pneumonia in humans

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M. Buač et al. / Research in Veterinary Science 106 (2016) 135–142

and experimental animals (Quinton and Mizgerd, 2015), but also in calves (Mosier, 2014). In our previous work we showed that calf bronchopneumonia is accompanied with an increased level of PEG precipitable circulating immune complexes (CIC) (Fratrić et al., 2012). We have also showed that IgG from CIC of diseased calves express more galactose and sialic acid than IgG from CIC of healthy calves. Such IgG glycosylation pattern could influence IgG-FcγR binding (Kapur et al., 2014) and we have assumed that it could also influence effector functions of these IgG containing CIC. Therefore in this study, with in vitro tests, we analyzed if CIC of three month old calves with bronchopneumonia were able to modulate function (adhesiveness, ROS and NO generation, viability and proliferation) of quiescent peripheral blood mononuclear cells (MNCs) and granulocytes of healthy calves. Given that age influences the effectiveness of the immune response in cattle (Chase et al., 2008), we also analyzed if CIC have a different impact on functional responses of blood leukocytes of young, three month old calves, and adult cows, at 30 days after parturition. 2. Material and methods 2.1. Animals In this study, 12 three months old Holstein-Friesian calves were donors of CIC. Calves were from a commercial farm owned by PKB Corporation (Padinska Skela, Belgrade, Serbia). The use of animals was approved by the Ethical Committee of the Faculty of Veterinary Medicine, University of Belgrade in accordance with the National Regulation on Animal Welfare. All calves were clinically examined by a veterinarian. The calves were classified according to signs of respiratory disease as healthy (n = 6) or diseased (n = 6). Healthy animals had body temperature b 39.5 °C, respiratory rate b 40 min−1, no nasal discharge, no coughing and normal respiratory sounds. The overall clinical score for diseased calves was calculated according to McGuirk (2005). The scoring system is expressed in numbers from 0 to 3 (0: normal, 1: mild, 2: moderate, 3: severe). Total respiratory score b 4 is normal; =4 is to be monitored; and N4 indicates the need for treatment. Calves were considered diseased when they scored 6 or more and presented two or more clinical signs of respiratory disease. Diseased animals had at least three of the following clinical signs: body temperature ≥ 39.5 °C, respiratory rate ≥ 45 min− 1, nasal discharge, coughing or increased respiratory sounds. Calves' nasal swab cultures are representative of their lung isolates (DeRosa et al., 2000) and in order to identify the bacteria and ensure adequate antibiotic therapy, deep nasal swabs were taken. Tests were done by applying conventional bacteriological methods and an automatic identification system, BBL Crystal 134 Enteric/nonfermenter ID kit (Becton Dickinson GmbH, Heidelberg, Germany), at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Belgrade. All samples from diseased calves were positive for P. multocida, while other pathogenic bacteria and fungi were not isolated. In nasal swab specimens of healthy calves no pathogenic bacteria or fungi were isolated. The analysis of basic hematological parameters of peripheral blood of these calves was done at the Faculty of Veterinary Medicine, Belgrade using the Hematology Analyzer 901,062 (Diatron, Arcus, GmbH, Wien, Austria). The obtained results are shown in Table 1. As donors of peripheral blood leukocytes, used as responders in the below listed in vitro tests, 5 three months old healthy calves and 5 healthy cows at 30 days after parturition, were included in this study. These calves and cows were also from the same above-mentioned commercial farm. 2.2. Polyethylene glycol (PEG) precipitation assay CIC were isolated by PEG precipitation assay (Fratrić et al., 2006). Blood samples from diseased calves, prior to treatment with antibiotics, and healthy calves were collected via jugular vein puncture. Blood

Table 1 Basic hematological indices of peripheral blood of healthy calves and calves with bronchopneumonia.

Erythrocytes (1012/l) Hemoglobin (g/l) Hematocrit (%) Leukocytes (109/l) Lymphocytes (109/l) Monocytes (109/l) Granulocytes (109/l) Lymphocytes (%) Monocytes (%) Granulocytes (%) Gr/Ly ratioa Platelets (109/l)

Healthy calves (n = 6)

Calves with bronchopneumonia (n = 6)

9.9 ± 0.4 87 ± 6 24.8 ± 1.8 7.0 ± 1.2 6.2 ± 0.8 0.3 ± 0.5 2.2 ± 1.6 81.6 ± 3.9 0.8 ± 0.2 17.7 ± 3.9 0.3 ± 0.2 420 ± 96

11.1 ± 0.3⁎⁎ 102 ± 3⁎⁎ 28.5 ± 1.3⁎⁎ 14.8 ± 3.7⁎⁎ 6.9 ± 1.0 0.4 ± 0.5 7.6 ± 3.8⁎ 49.8 ± 15.7⁎⁎ 2.3 ± 3.2 47.9 ± 15.7⁎⁎ 1.0 ± 0.6⁎⁎ 390 ± 103

Data are mean ± SD. The significant difference between healthy and diseased calves: ⁎P b 0.05; ⁎⁎P b 0.01. a Gr/Ly — granulocytes/lymphocytes.

serum was separated after spontaneous coagulation and centrifugation. In 2 ml of fresh serum, 6 ml of 4.5% (w/v) PEG (MW 6000) in 100 mM sodium borate buffer pH 8.3–8.5 (all from Sigma Taufkirchen, Germany) was added. After 2 h of incubation at 4 °C, samples were centrifuged for 30 min at 2060 ×g, at 4 °C. The precipitated proteins were redissolved in 2 ml of sterile PBS (0.8% NaCl, 10 mM sodiumphosphate, pH 7.2–7.4). Optical densities at 350 nm (OD350) of the redissolved PEG precipitates were measured on Ultrospec 3300pro spectrophotometer (Amersham Bioscience, Uppsala Sweden). Aliquots of the PEG precipitates were stored at −20 °C pending analysis. 2.3. Concentration of γ globulin in sera and CIC Agarose gel electrophoresis of total serum proteins and PEG precipitated serum proteins was performed according to the procedure of Johansson (1972). The relative content of γ globulins (percentage) was quantified by densitometry using ImageMaster Total- Lab v1.11 software (Amersham Pharmacia Biotech, Uppsala Sweden). Concentration of γ globulins was calculated based on total protein concentration determined by the BCA protein assay kit (Pierce, Rockford IL, USA). 2.4. Isolation of peripheral blood leukocytes Blood of healthy calves and cows was drawn from the jugular vein into 50 ml sterile tubes containing 5 ml 3.8% sodium-citrate. Separation of MNCs was performed by whole blood centrifugation over FicollHypaque (1.077) density gradient (PAA Laboratories, Pasching, Austria), for 35 min, at 400 ×g at 4 °C. The interface MNCs were collected, washed with PBS and resuspended in a complete cell culture medium (CM), RPMI 1640 (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (PAA Laboratories), and penicillin/streptomycin (PAA Laboratories). Granulocytes from the lower layer were purified by lysis of erythrocytes in isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4; all from AppliChem GmbH, Darmstadt, Germany). Granulocytes were then washed in PBS and resuspended in the CM. 2.5. Measurement of cell adhesion to polystyrene Adhesion of MNCs and granulocytes to plastic (polystyrene) was assessed with the assay described by Vlaški et al. (2004). Peripheral blood granulocytes or MNCs (5 × 105 cells in 100 μl CM /well) were plated in 96-well flat bottom plates with 10 μL of CIC and incubated for 60 min. Spontaneous adhesion was determined in control cultures of cells incubated in CM only. Cultures of cells stimulated with PMA (phorbol 12-myristate 13-acetate) (50 ng/ml) (Sigma-Aldrich, St. Louis, MO) were the assay positive control. After incubation, non-


adherent cells were carefully removed and the cells adhering to the plastic surface were washed with saline, fixed with methanol, and stained with 0.3% crystal violet. After staining, the plates were washed in water, and air-dried. The precipitated dye was dissolved in 33% acetic acid, and the OD540 was measured in an ELISA 96-well plate reader (Multiscan Plus Reader, Labsystem, Finland). The results were given as the relative level of cell adhesion where the OD540 level of control cultures was set as 100%.

2.6. NBT reduction assay The synthesis of ROS in MNCs and granulocytes was evaluated by a cytochemical assay for the respiratory burst (Vlaški et al., 2004) measured by the intracellular reduction of nitroblue tetrazolium salt (NBT) (Merck, Germany). Cells were plated in 96-well flat bottom plates (5 × 105 cells in 100 μl CM/well) and incubated for 5 min with 10 μL of CIC. The spontaneous NBT reduction was determined in control cultures, consisting of cells incubated in CM only. Cultures of cells stimulated with PMA (50 ng/ml) were the positive controls. After 5 min incubation, NBT (5 mg/ml; 10 μl/well) was added and the cells were additionally incubated for 30 min at 37 °C. Formazan produced by the cells was extracted overnight in 10% SDS/0.1 N HCl at 37 °C and OD540 was measured on a 96-well plate reader. The results were given as relative levels of ROS synthesis where the OD540 level of control cultures was set as 100%.

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2.9. CFSE (5-(and 6)-aarboxyfluorescein diacetate, succinimidyl ester) assay Proliferation of MNCs was determined using the CFSE assay. In brief: MNCs (2.5 x106 /ml PBS containing 5% FBS) were labeled with 5 μM CFSE dye (eBioscience, San Diego, CA). CFSE stained MNCs, unstimulated or PHA (2.5 μg/ml) stimulated (5 × 105 cells/500 μl/well; 24 well-plates), were cultured with or without 50 μl of CIC. The cultures of unstimulated MNCs seeded without CIC were used as controls. After 6 days in culture, the cells were analyzed on a CyFlow® SL flow cytometer. Proliferation index for every sample was calculated as described by Parish and Warren (2002) and the proliferation index of control cultures was set as 100%. 2.10. Propidium iodide (PI) staining For the estimating of the effect of CIC on apoptosis/necrosis, MNCs cells were subjected to flow cytometry analysis after PI staining. Briefly, after 48 h (unstimulated cells) or 72 h (PHA stimulated cells) in culture, MNCs were washed and stained (1 × 105 cells in 100 μl) with PI (5 μg/ ml) (Beckman Coulter International SA, Nyon, Switzerland). After 15 min of incubation, the cells were analyzed on a CyFlow® SL flow cytometer (Partec, Münster, Germany) using FlowMax 2.4 software (Partec, Münster, Germany). The cells having PI fluorescence higher than the cut-off value of non-specific fluorescence were declared as positive. The percentage of positive cells was expressed as the ratio of labeled to total cell number.

2.7. Measurement of nitric oxide production

2.11. Statistical analysis

MNCs or granulocytes were plated in 96-well plates (5 × 105 cells in 100 μl CM/well) and cultivated for 48 h with 10 μL of CIC. Control cultures were the cells incubated in CM or with 10 μg/ml LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich, SAD). Production of nitric oxide, quantified by the accumulation of nitrite in the culture medium, was determined spectrophotometrically using the Griess reaction with sodium nitrite as standard (Green et al., 1982). The detection limit of the assay was 3 μM of nitrite.

The statistical significance of differences between two groups was determined by the two-tailed Mann-Whitney U test, using interactive software on http://www.socscistatisics.com webpage. Differences with p-values of b 0.05 were considered significant. Correlation between the OD350 values of PEG precipitable CIC and concentrations of serum γ globulins were determined by Spearman rank correlation test, using interactive software on http://vassarstats.net/corr_rank.html webpage. 3. Results

2.8. MTT assay Viability/metabolic activity of the peripheral blood cells was analyzed by a quantitative colorimetric MTT (3-(4,5-dimethyl-thiazol2yl)-2,5 diphenyltetrazolium bromide, ICN Biomedicals, Ohio) assay as described by Vlaški et al. (2004). First, the MTT assay was used for assaying cell viability (number) of unstimulated MNCs and granulocytes. The cells were plated in 96-well flat-bottom plates (5 × 105 cells in 100 μl CM /well) and cultured with 10 μL of CIC for 48 h at 37 °C. Cultures of MNCs seeded in CM were used as the control. MTT (5 mg/ml, 10 μl/well) was added immediately after seeding, or after 48 h in culture, and the cultures were incubated for 3 h at 37 °C in a humidified atmosphere. Formazan produced by the cells was dissolved during overnight incubation in 10%SDS/0.1 N HCl and its absorbance was measured at 540 nm (OD540) in the 96-well plate reader. The results were presented as relative viability, where the OD540 levels of fresh MNCs were set as 100%. Furthermore, the MTT assay was used for estimating the effects of CIC on the viability of phytohaemagglutinin (PHA) stimulated MNCs. The cells (5 × 105 cells/100 μl/well) were plated with 10 μl of CIC and stimulated with 2.5 μg/ml PHA (INEP, Zemun, Serbia). Cultures of PHA-stimulated MNCs seeded without CIC were used as controls. After 72 h, MTT was added and the level of produced formazan was determined. The results are presented as relative viability where the viability of PHA stimulated MNCs without CIC was set as 100%.

3.1. The level of CIC and CIC's γ globulins in calves with bronchopneumonia The OD350 level of the PEG precipitated CIC of calves with bronchopneumonia was 2.8 times higher than the one of healthy calves (Table 2). The concentration of CIC's and serum γ globulins was higher Table 2 The CIC level and γ globulins in CIC and in sera of healthy calves and calves with bronchopneumonia. Healthy calves (n = 6) CIC level (OD350)

0.217 ± 0.055⁎⁎

0.077 ± 0.027 %a

CIC proteins CIC γ globulins Serum proteins Serum γ globulins

Calves with bronchopneumonia (n = 6)

18 ± 3 22 ± 7

mg/ml 1.9 ± 0.2 0.3 ± 0.1 83 ± 7 18 ± 7

%a 32 ± 13b 34 ± 6b

mg/ml 2.8 ± 0.5⁎⁎ 1.0 ± 0.6 ⁎⁎ 111 ± 22⁎ 42 ± 17⁎

CIC were isolated by PEG precipitation. Data are mean ± SD. a Percentage of total (CIC or serum) proteins; b The significant difference in the percentages of γ globulins between healthy and diseased calves; ⁎P b 0.05. ⁎ The significant difference in the CIC level and concentrations of total proteins and γ globulins between healthy and diseased calves; ⁎P b 0.05; ⁎⁎P b 0.01.

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in diseased animals (Table 2). However, the percentage of PEG precipitated serum γ globulins was almost equal in healthy and diseased calves. Thus, PEG precipitated 2.0 ± 0.6% and 2.3 ± 0.8% of serum γ globulins in healthy and diseased animals, respectively. The level of CIC did not significantly correlate with the concentration of serum γ globulins in any of the analyzed groups (rs values for healthy and diseased animals were −0.09, and −0.58, respectively). 3.2. Modulatory effects of CIC of healthy calves and calves with bronchopneumonia on functions of healthy cattle peripheral blood leukocytes: in vitro analysis The results given in Fig. 1 showed that CIC modulated adhesion of MNCs and granulocytes to plastic (polystyrene). Spontaneous adhesion of calves' MNCs was weak. PMA and CIC of diseased calves increased it by 75% and 25% respectively, while CIC of healthy calves were of no influence. Adhesion of cows' MNCs was not affected by PMA and CIC of diseased calves, and was even inhibited (30%) in the presence of CIC of healthy animals. In contrast to MNCs, granulocytes of both calves and cows strongly increased adhesion (240% and 1400%) in response to PMA stimulation. CIC of both healthy and diseased calves slightly (approximately 20%) stimulated adhesion of calves' granulocytes. CIC of diseased calves strongly stimulated the adhesion of granulocytes of adult animals (360%), while CIC of healthy calves were ineffective as stimulators of adhesion. In this work, the respiratory burst activity, i.e. synthesis of ROS, in peripheral blood leukocytes upon PMA and CIC stimulation was evaluated by the NBT test (Fig. 2). All analyzed cells were strong responders to PMA stimulation. Calves' MNCs and granulocytes increased ROS synthesis by 440% and 500%, while cows' MNCs and granulocytes increased it by 1460% and 3000%. CIC were less potent inducers of ROS synthesis than PMA. A mild to moderate increase of CIC stimulated ROS synthesis in calves' MNCs and granulocytes, and in cows' MNCs (80%, 30% and 110% respectively) was detected for both CIC of healthy and diseased animals. Cows' granulocytes were much better responders to CIC. They increased ROS synthesis in response to CIC of healthy calves up to 300% and to CIC of diseased calves up to 430%. However, the difference between these values was not significant due to the high level of individual variations. Nitrites in supernatants of cultures of peripheral blood MNCs and granulocytes, grown with the CIC, were below detection levels (b3 μM). The same was detected when the cells grown in CM or were stimulated with LPS (data not shown). The impact of CIC on the viability of peripheral blood granulocytes of healthy cattle was estimated by MTT assay (Fig. 3). The results showed that the spontaneous viability of calves' granulocytes after 48 h in

cultures decreased by 60%, and that CIC additionally reduced it. The viability of cows' granulocytes in the short-term culture was reduced by 20%, and CIC also additionally reduced it. The effect of CIC of diseased calves was more pronounced (50% inhibition) then the effect of those of healthy calves (40% inhibition). The viability of unstimulated MNCs in 48 h culture was also estimated by the MTT test (Fig. 4A). The viability of cows' MNC spontaneously decreased by 20% and CIC did not additionally influence it (Fig. 4A). The viability of unstimulated calves' MNCs in 48 h cultures spontaneously decreased by 15% and the presence of CIC additionally decreased it. Again, the influence of CIC of diseased calves was more pronounced (inhibition by 30%) than those of healthy calves (inhibition by 25%). The effect of CIC was very strong in the cultures of mitogen (PHA) stimulated MNCs, where CIC healthy and diseased calves reduced the viability of both calves' and cows' cells by 50% (Fig. 4B). The modulatory effect of CIC on the proliferation of calves' MNCs was further-on studied by CFSE staining. The results (Fig. 5A and B) showed that proliferation of unstimulated MNCs was two times higher in the presence of CIC, and the recorded proliferation index was even higher than of MNCs stimulated with PHA. However, CIC of both healthy and diseased calves induced only a mild inhibition of proliferation of PHA stimulated MNCs. CIC of healthy calves increased the number of PI+ cells by 40% and 70% in cultures of unstimulated and PHA stimulated MNCs, whereas CIC of diseased animals increased it by 70% and 110% (Fig. 5C). 4. Discussion The formation of immune complexes is a normal physiological event in the processes of elimination of antigens. They act as immune modulators directing either a pro- or anti-inflammatory immune response, depending on the specific immunological context at the site of inflammation (Bournazos et al., 2015). Previously we showed that calf bronchopneumonia is accompanied with an increased level of CIC and that their IgG have the glycosylation profile different from those of healthy calves' CIC (Fratrić et al., 2012). The role of these IgG containing CIC are not known. In this study we determined if CIC of calves with bronchopneumonia have a capacity to modulate function of peripheral blood granulocytes and MNCs of healthy cattle. We chose this approach knowing that leukocytes of healthy animals circulate in the vasculature in a passive, quiescent state, and only in the course of induced lung inflammation they are recruited to the site of inflammation, where become fully activated and express their effector functions (Gao et al., 2006). Adhesion of peripheral blood leukocytes to inflammatory activated endothelial cells is the first step in their activation, and it is followed

Fig. 1. CIC of calves with bronchopneumonia modulate the adhesion of peripheral blood leukocytes. The measured OD540 values of CIC treated granulocytes and MNCs were normalized to the control ones (without CIC). Results are mean ± SD. (**) and (*) — The significant difference between the CIC stimulated and control cells: P b 0.01 and P b 0.05. (#) — Statistically significant difference in the effects of CIC of healthy and diseased calves: P b 0.05.


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Fig. 2. CIC of calves with bronchopneumonia induce ROS synthesis in peripheral blood leukocytes. ROS synthesis was estimated by NBT test. The OD540 values of the CIC treated cell cultures were normalized to the control ones (without CIC). Results are mean ± SD. (**) and (*) — Statistically significant difference between the CIC stimulated and control cells: P b 0.01 and P b 0.05.

by ROS generation. The ability of cows' and (to a less extent) calves' granulocytes to increase their adhesiveness and ROS generation upon PMA stimulation showed us that they are functionally competent cells, able to, in response to an appropriate stimulation, became activated and to synthesize ROS. Research conducted on rodent models showed that IgG immune complex mediated lung inflammation is neutrophil dependent (Oldham et al., 1988). According to this, we showed that cows' granulocytes strongly increase their adhesiveness and generated a considerable amount of ROS in response to CIC stimulation. Additionally, these granulocytes were unique among the analyzed cells in their superior response to CIC of diseased calves. Higuchi and Nagahata (1998) reported that heat aggregated IgG (in vitro analogue of immune complexes) induced a significantly lower superoxide production in calves' neutrophils than in cows' ones. We associated the inability of calves' granulocytes to increase the adhesiveness and ROS synthesis in response to CIC stimulation with the functional immaturity of immune cells of young animals (Chase et al., 2008). In contrast to the adhesion of granulocytes, adhesion of MNCs was not strongly stimulated either with PMA or CIC. Regarding MNCs' ROS synthesis, PMA was strong, whereas CIC were weak inducers. Bovine MNCs are composed of many cell types (monocytes, T cells, B cells, NK cells, dendritic cells), each consisting of its own subpopulations, existing in many different activation states (Chattha et al., 2009; Hussen et al., 2013; Maślanka, 2014). Humans and rodents MNCs express both activating FcγR (FcγRI and FcγRIIa on monocytes, and FcγRIIc, and FcγRIIIa

Fig. 3. CIC of calves with bronchopneumonia reduce the viability of peripheral blood granulocytes. The viability of granulocytes in 48 h cultures was determined by MTT test. The OD540 values were normalized to fresh granulocytes. Results are mean ± SD. (⁎⁎) — The significant difference in the viability of the CIC stimulated cows' granulocytes versus the control cells grown in CM: P b 0.01; (##) — Statistically significant difference in the effect of CIC from healthy and diseased calves: P b 0.01.

on monocytes and NK cells) and inhibitory FcγR (FcγRIIc on B cells and subpopulation of monocytes) (Kapur et al., 2014; Bournazos et al., 2015). The influence of IgG containing immune complexes and FcγR intracellular signaling on lung inflammation is confirmed in rodent models which showed that FcγRI and FcγRIII have a pro-inflammatory role, whereas FcγRII acts anti-inflammatory (Sarma and Ward, 2007). The resulting immune-complexes mediated response is largely

Fig. 4. CIC of calves with bronchopneumonia reduce the viability of both unstimulated and PHA stimulated peripheral blood MNCs. The viability of MNCs in short term cultures was determined by MTT test. (A) The spontaneous viability of MNCs in 48 h cultures. The OD540 values were normalized to fresh MNCs. (B) The viability of PHA (2.5 μg/ml) stimulated MNCs in 72 h cultures. The OD540 values were normalized to control, PHA stimulated cells. Results are mean ± SD. (***) and (**) — The significant difference between the viability of the CIC stimulated and control MNCs grown in CM: P b 0.001 and P b 0.01; (#) — Statistically significant difference in the effect of CIC of healthy and diseased calves: P b 0.05.

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Fig. 5. CIC of calves with bronchopneumonia modulate proliferation and apoptosis of both unstimulated and PHA stimulated peripheral blood MNCs. The MNCs proliferation was determined by CFSE assay. (A) CFSE profile of unstimulated (1) and PHA stimulated (2) calves' MNCs cultured with or without CIC. Grey plot: Control cultures — MNCs w/o the CIC; blue line: MNCs cultured with CIC of healthy calves; red line: MNCs cultured with CIC of calves with bronchopneumonia. (B) Proliferation index of spontaneous and PHA (2.5 μg/ml) stimulated of MNCs. (C) Percentage of PI+ apoptotic/necrotic MNCs. The results are of one representative experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

determined by balancing intracellular signals transduced by different FcγR (Bournazos et al., 2015). In cattle FcγR homologous to human FcγRI, FcγRII, and FcγRIII are described. (Kacskovics, 2004). Differently from humans and experimental animals, cellular expression and functions of bovine FcγR have not been extensively studied. We can only speculate that a convergence of signaling pathways from many different FcγRs resulted in the absence of MNCs adhesiveness and mild ROS synthesis in response to the CIC stimulation. Besides, reported agedifferences in the expression of FcγRII on bovine peripheral blood MNCs (Chattha et al., 2009, 2010) could partially explain the different effect of CIC on calves' and cows' cells.

NO is a potent mediator of pulmonary inflammation/injuries caused by immune complexes (Mulligan et al., 1991), and immune complexes induce its synthesis in resident lung cells (Warner et al., 1995). Although NO synthesis can be induced by different stimuli in bovine peripheral blood leukocytes (Goff et al., 1996), we did not observe a detectable NO synthesis in CIC stimulated cultures of cattle's peripheral blood leukocytes. This finding could be in accordance with the result from rodent models of lung injury, which showed that lung resident cells, but not recruited blood leukocytes, are the main source of NO in inflamed lung tissue (Ward, 2010).


Lung inflammation can be regulated by modulation of the lifespan of lung hematopoietic and non-hematopoietic cells (Gao et al., 2006). In acute infection and/or inflammation the lifespan of neutrophils is prolonged in order to combat the inflammation and it can be controlled by induction of apoptosis or necrosis. According to Haslett (1999) elimination of granulocytes by apoptosis is one of the mechanisms for the attenuation of lung inflammation. Gamberale et al. (1998) showed that immune complexes modulate granulocytes apoptosis, but the effect is dependent on their molecular structure. Precipitating immune complexes stimulated apoptosis of granulocytes, whereas soluble immune complexes delayed apoptosis. Haslett (1999) reported that necrosis of granulocytes and subsequent release of their granules contents intensifies the inflammation. However, the precise role of apoptosis and necrosis of neutrophils in lung inflammation is not resolved yet. Thus Hart et al. (2004) showed that immune-complexes through FcγRIIA binding on apoptotic neutrophils increased their phagocytosis by macrophages and stimulated the release of pro-inflammatory IL-1 and IL6. However, Miles et al. (2009) showed that necrotic and apoptotic neutrophils secreted anti-inflammatory factors, which inhibit secretion of pro-inflammatory cytokines without affecting microbe destruction. In this study, we showed that CIC reduced the viability in short-term cultures of both calves' and cows' granulocytes, with the most prominent effect showed by CIC of diseased calves on cows' granulocytes. If the observed reduction of granulocytes' viability (i.e. the lifespan) induced by CIC are protective or damaging is not clear. PEG precipitates only high molecular weight CIC (Poulton et al., 1983) which are in humans, associated with less severe forms of pneumonia, thus might have a protective role (Mizutani and Mizutani, 1986; Mellencamp et al., 1987). Based on these data we speculated that the observed CIC-reduced viability of granulocytes might have a protective role in calf bronchopneumonia. In mitogen (PHA) stimulated cultures, CIC strongly reduced the viability of calves' MNCs. Results of CFSE showed that CIC strongly stimulated the spontaneous proliferation of MNCs but only slightly reduced PHA-stimulated proliferation. Besides, in the presence of CIC, the percentage of apoptotic cells in both, unstimulated or PHAstimulated MNCs cultures, increase. These multiple and seemingly opposite effects of CIC on the MNCs are a feature of many immune modulators. As we noted above, bovine MNCs are composed of many cell types expressing different FcγR. In such a complex environment, immune complexes in accordance with local microenvironment signals trigger different cellular responses. They should result in an optimally balanced immune response, which can be either stimulatory or inhibitory depending on the specific needs of the organism. That is why we assumed that the greatly increased proliferation of non-stimulated MNCs (predominantly lymphocytes) might be important in the early stages of the disease when the number of antigen specific lymphocytes should increase. On the other hand, the greatly reduced viability of mitogen stimulated MNCs (resulting from increased apoptosis and reduced proliferation of mitogen stimulated cells) might play a role in resolving lung inflammation leading to the elimination of immune complexes activated lymphocytes. The observed more prominent effects of CIC of diseased calves cannot be related exclusively to specific structural properties of CIC's IgG (Fratrić et al., 2012). Molecules of the microbial pathogen also act as powerful modulators of many functions of bovine immune cells (Shewen and Wilkie, 1982; Sun et al., 2000; Cudd et al., 2001; Mulongo et al., 2013; Wang et al., 2013). Therefore, P. multocida molecules, as constituents of the CIC, could be responsible for some of the observed effects. Besides, some autoantigens can form immune complexes of importance for lung diseases. Thus, immune complexes of IL-8 and anti-IL-8 autoantibodies mediate lung inflammation by activating endothelial cells via IgG receptors (Krupa et al., 2009). Molecular constituents of the analyzed CIC which are of importance for the observed in vitro effects remain to be determined.

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