Медицина/7. Клиническая медицина

Nikolenko O.Y., Lygina J.A., Nikolenko V.Y.

Donetsk national medical university of M.Gorkogo, Donetsk, Ukraine

PECULIARITIES OF PHAGOCYTIC ACTIVITY OF NEUTROPHILS IN RATS WITH A MODEL OF COLINET-CAPLAN’S SYNDROME

Current understanding of the pathogenesis of silicosis as well as rheumatoid arthritis allows to state that Colinet-Caplan’s syndrome is the only disease that begins with the development of pneumoconiosis under the influence of free silicon dioxide, then there appear immunological disorders that lead to rheumatoid arthritis, which, in its turn, is accompanied by the development of lung rheumatoid nodules in the interstitium. The formation of nodules is caused by the presence of dust particles in alveolar macrophages [2, 3].

Autoimmune diseases are accompanied by deviations from the normal course of apoptosis of the human organism. Superfluous accumulation of apoptotic cells, due to violation of their utilization by phagocytes involving complement, leads to changes in the spectrum of the dominant cytokines from inflammatory IL-1 and TNF-alpha to the anti-inflammatory IL-4, IL-10, which promotes long-term maintenance of the Th2-type immune response. [1]

In recent years it has been demonstrated that biological processes of organism protection including phagocytosis are directly related to the reactive oxygen species formation in the cells. With pulmonary dust disease "respiratory burst" is able not only to destroy coniophages but also run an autoimmune mechanisms. The formation of reactive oxygen species and development in coniophages of energy deficient status and intracellular hypoxia determines the inefficiency of antimicrobial immunity, especially in the interaction of industrial fibrogenic dust high in silica, which has the properties of immunological adjuvants [4].

Material and methods. In experiment two such groups of rats (male line "Wistar" weighing 200-250 g.) are used: Group 1 - healthy animals (25 rats), Group 2 - animals with modeling Colinet-Caplan’s syndrome (the root of the tail of the animal was injected with complete Freund’s adjuvant). On the 7th day the animal was injected 1 ml of a suspension of coal dust at the rate of 50 mg dust per rat. On the15th and 34th days 0.5 ml of complete Freund's adjuvant was injected to the root of a rat tail. After 29 and 40 days cytotoxic agents were administered - azathioprine in a dose of 50mg/kg, and from 16th to 29th days and from 43th to 49th days Methyluracilum was administered in a dose 0.2 g / kg). Research took 49 days. In 10 days after the last injection of Methyluracilum animals were sacrificed under ether anesthesia.

During the decapitation of animals blood sampling was carried out, smears were prepared, fixed in methanol, then stained by Romanovsky.

Interleukins 6 and 10 were determined in serum by solid-phase enzyme immunoassay with the use of kits from PLC "Cytokine" St. Petersburg (Russian Federation). In a set of "ELISA IL-10" "sandwich" - variant of solid-ELISA was used.

To determine the phagocytic activity of peripheral blood neutrophils of patients' daily test culture aureus strain 209 was used. During microscopic studies (at least 200 cells were counted) calculations of phagocytic index after 30 and 90 min were carried out.

Oxygen-dependent metabolism of neutrophils was studied by using a test with nitroblue tetrazolium (NBT). In case of contact with activated neutrophils, NBT updated in dyformazan, which in the form of pellets, insoluble in water and organic solvents, was deposited within and on the surface of cells. Quantity of dyformazan that was deposited was the criterion of the intensity of the reaction. After microscopy the percentage of neutrophils containing dyformazan and neutrophil activation index (NAI) was calculated.

For processing the results of studies using correlation and regression analysis techniques, assessing the main value () and their standard errors (S), Student's criterion (S), the median criterion (Mt), Mann-Whitney (MW) reliability of statistical parameters (p) on the computer SAMSUNG (R 20) using licensed package «Statistica 5,5» (Start Soft Rus).

Research results. One of the parts of humoral and cellular immunity is phagocytic link. Comparing the indicators of the phagocytic activity of neutrophils after 30 min in animals experimental group it was amounted (18,72 ± 0,62)%, differing from control (23.43 ± 0.04)% (MW = 6.06, p<0.001, Mt = 50.00, p<0.001, S = 7.56, p<0.001). Phagocytic number in animal model with Colinet-Caplan’s syndrome was (5.32 ± 0.23) after 30 minutes, differing from controls (6.73 ± 0.07) (MW = 4.07, p<0.001, Mt = 18.00, p<0.001, S = 5.77, p<0.001), phagocytic activity in infected animals after 30 minutes was significantly lower (18.72 ± 0.62)% compared with control rats (23.43 ± 0.04)% (MW = 6.06, p<0.001, Mt = 50.00, p<0.001, S = 7.56, p<0.001), phagocytic activity in infected animals was (18.96 ± 0.61)% after 90 minutes, which differed from the control (23.66 ± 0.04)% (MW = 6.01, p<0.001, Mt = 46.15, p<0.001, S = 7.73, p<0.001 ) and phagocytic number in model animals was (5.46 ± 0.45) after 90 minutes, differing from controls (6.91 ± 0.07) (MW = 1.49, p = 0.135, Mt = 0.73 , p = 0.39, S = 3.46, p<0.001) only in accordance with the Student's criterion.

These data show that the animal at the model of Colinet-Caplan’s syndrome has reduced rates of phagocytosis of neutrophils. This could indicate at the involvement of these cells in immunopathological processes (Fig. 1).

Figure 1. Phagocytic activity of neutrophils by phagocytosis 209 in rat model of Colinet-Caplan’s syndrome and in control.

Note: 1 - phagocytic activity of neutrophils after 30 minutes (%) 2 - phagocytic number after 30 minutes (staphylococci), 3 - phagocytic activity of neutrophils after 90 min (%) 4 - phagocytic number in 90 minutes (staphylococci).

Groups: - model of Colinet-Caplan’s syndrome; - control

These data show that the animal at the model of Colinet-Caplan’s syndrome has reduced rates of phagocytosis of neutrophils.

Development of the disease by type of Colinet-Caplan’s syndrome influenced the decrease of the phagocytic activity of neutrophils with staphylococcus 209 after 30 minutes (KW = 37.01, p<0.001), number of phagocytes after 30 minutes (KW = 16.58, p<0.001), phagocytic activity of neutrophils with staphylococcus 209 after 90 minutes (KW = 36.37, p<0.001).

Comparing the ability of neutrophils to oxygen-dependent metabolism by NBT-test revealed that the animal model is much smaller (37.80 ± 3.76)% compared with controls (82.96 ± 3.41)% (MW = 5.53, p<0.001, Mt = 35.28, p<0.001, S = 8.89, p<0.001), respectively.

Neutrophils activation index without stimulation was on sick animals (0.61 ± 0.06), differing from controls (1.87 ± 0.10) (MW = 5.68, p<0.001, Mt = 42.32, p<0.001, S = 10.64, p<0.001), respectively. Thus, with the Colinet-Caplan’s syndrome phagocytes have low reserve activity of intra-cellular enzymes that can lead to loss of neutrophils during their activation and deficiency of phagocytic component of the immune system.

Development of the disease by type of the Colinet-Caplan’s syndrome influenced the reduced ability of neutrophils to oxygen-dependent metabolism by NBT-test (KW = 30.60, p<0.001), index of neutrophils activation without stimulation (KW = 32.22, p<0.001).

Comparing the quantity of interleukins 6, 10 among animals with model of the Colinet-Caplan’s syndrome and controls their content was greatly differ.

The level of IL-6 in the control group (43.27 ± 2.57) pg / ml was lower than in rats with the model of Colinet-Caplan’s syndrome (51.26 ± 1.50) pg / ml (MW = 2.05, p<0.001, Mt = 2.00, p = 0.16, S = 2.69, p = 0.01), respectively. Elevated levels of IL-6 may lead to the stimulation of proliferation of fibroblasts and formation of fibrous inflammation. Also this cytokine enhances proliferation and differentiation of B-cells, which may lead to decrease of the phagocytic function of neutrophils. We have received credible evidence of concentrations increase of IL-10 in blood serum in rats with the model of Colinet-Caplan’s syndrome (57.13 ± 1.56) pg / ml compared with the control group (49.44 ± 2.55) pg / ml (MW = 2.18, p<0.001, Mt = 3.92, p = 0.05, S = 2.57, p = 0.013), respectively. Elevated levels of inflammatory cytokines IL-10 can sustain chronic inflammation in the Colinet-Caplan’s syndrome. Also this cytokine enhances proliferation and differentiation of B-cells, which may lead to a decrease of the phagocytic function of neutrophils.

Conclusions.

1. In the study of rats’ blood with the model of the Colinet-Caplan’s syndrome decrease of the phagocytic activity of neutrophils in response to phagocytosis of staphylococcus strain 209 and the NBT test was found.

2. In a rat with the model of Colinet-Caplan’s syndrome in serum increasing of anti-inflammatory interleukins 6 and 10 was detected that can prove of the maintenance of chronic inflammation.

3. Knowledge of immunopathogenesis of the Colinet-Caplan’s syndrome model in rats can be used to develop new treatments.

References:

1. Murray, John F. (2010). Murray and Nadel's textbook of respiratory medicine. (5th ed.). Philadelphia, PA: Saunders/Elsevier. 2400 Pages.

2. Orlowski E., De Cremoux H., Pairon J.C., Merlet C., Bignon J., Brochard P. Exposition à la silice et maladies systémiques. Arch Mal Prof 1990 ; 51 : 591-93.

3. Rheumatoid pneumoconiosis (Caplan's syndrome) /J. Schreiber, D. Koschelb, J. Kekowc, N. Waldburga, A. Goetted, R. // Mergete European Journal of Internal Medicine Volume 21, Issue 3, June 2010, Pages 168–172

4. Multiwalled Carbon Nanotubes Induce a Fibrogenic Response by Stimulating Reactive Oxygen Species Production, Activating NF-κB Signaling, and Promoting Fibroblast-to-Myofibroblast Transformation / Xiaoqing He , Shih-Houng Young , Diane Schwegler-Berry , William P. Chisholm , Joseph E. Fernback , and Qiang Ma // Chem. Res. Toxicol., 2011, 24 (12), pp 2237–2248.