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TEMPORAL DYNAMICS OF CYTOKINES IN THE BLOOD OF RATS WITH EXPERIMENTALLY INDUCED AUTOIMMUNE ENCEPHALOMYELITIS
Pozdniakova NV1, Turobov VI2, Garanina EE3, Ryabaya OA1, Biryukova YuK4, Minkevich NI2, Trubnikova EV5, Shevelev AB6, Kuznetsova TV7, Belyakova AV4, Udovichenko IP2>8E3
1 Blokhin Russian Cancer Research Center, Moscow, Russia
2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Pushchino branch
3 Kazan Federal University, Kazan, Russia
4 Chumakov Federal Center for Research and Development of Immunobiological Products, the Russian Academy of Sciences, Moscow, Russia
5 Kursk State University, Kursk, Russia
6 Emanuel Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow, Russia
7 Vavilov Institute of General Genetics of the Russian Academy of Sciences, Moscow, Russia
8 Pushchino State Institute of Natural Sciences, Pushchino, Russia
In this work we explore the temporal dynamics of cytokines in Dark Agouti rats with experimentally induced autoimmune encephalomyelitis (EAE). The main group consisted of 11 animals who were injected with 100 pl (per leg) of spinal cord homogenate obtained from random-bred rats and combined with incomplete Freund's adjuvant to the hind footpads. The control group included 7 animals who received 100 pl of normal saline mixed with incomplete Freund's adjuvant. Blood samples (500 pl) were collected daily, starting from day 1 through day 7. We ran a Bio-Plex-based multiplex cytokine assay on the samples using the Bio-Plex Pro Rat Cytokine 24-plex Assay kit. EAE in rats was shown to simulate progression of multiple sclerosis in humans in terms of temporal dynamics of lymphoproliferative and hematopoietic factors IL-1b, IL-2, IL-4, IL-5, IL-6, and IL-7. The studied model satisfactory imitates the dynamics of factors stimulating migration of lymphocytes, monocytes and other immune cells, including IL-17, RANTES (CCL-5) and MCP-1 (CCL-2) but excluding GRO/KC (CXCL1), which shows a different dynamics. The model also resembles patterns of human multiple sclerosis in terms of factors affecting cytotoxic and apoptotic reactions, including IFNy, IL-6 and IL-17, but excluding TNFa.
Keywords: multiple sclerosis, experimental autoimmune encephalomyelitis, myelin, immunization, multiplex cytokine assay
Acknowledgements: the authors thank Boris Shevelev for help in immunization of the animals.
Funding: this work was supported by the Ministry of Education and Science of the Russian Federation (Grant agreement 14.607.21.0133 dated October 27, 2015, ID RFMEFI60715X0133).
gg Correspondence should be addressed: Igor Udovichenko
Pr-t Nauki, d. 6, Puschino, Moscow oblast, Russia, 142290; iudovichenko1@yandex.ru
Received: 15.12.2017 Accepted: 20.12.2017
ВРЕМЕННАЯ ДИНАМИКА ЦИТОКИНОВ В КРОВИ ПРИ ЭКСПЕРИМЕНТАЛЬНОМ АУТОИММУННОМ ЭНЦЕФАЛОМИЕЛИТЕ У КРЫС
Н. В. Позднякова1, В. И. Туробов2, Е. Е. Гаранина3, О. А. Рябая1, Ю. К. Бирюкова4, Н. И. Минкевич2, Е. В. Трубникова5, А. Б. Шевелев6, Т. В. Кузнецова7, А. В. Белякова4, И. П. Удовиченко2*8 н
1 Национальный медицинский исследовательский центр онкологии имени Н. Н. Блохина, Москва
2 Филиал Института биоорганической химии имени академиков М. М. Шемякина и Ю. А. Овчинникова РАН, Пущино
3 Казанский (Приволжский) федеральный университет, Казань
4 Федеральный научный центр исследований и разработки иммунобиологических препаратов имени М. П. Чумакова РАН, Москва
5 Курский государственный университет, Курск
6 Институт биохимической физики имени Н. М. Эмануэля РАН, Москва
7 Институт общей генетики имени Н. И. Вавилова РАН, Москва
8 Пущинский государственный естественнонаучный институт, Пущино
Изучена динамика содержания цитокинов у крыс линии Dark Agouti с индуцированным экспериментальным аутоиммунным энцефаломиелитом (ЭАЭ). В экспериментальную группу включили 11 животных, которым в подушечки задних лап инъецировали гомогенат спинного мозга беспородных крыс, смешанный с неполным адъювантом Фрейнда. В контрольную группу включили 7 животных, которым в подушечки задних лап вводили по 100 мкл физиологического раствора, смешанного с неполным адъювантом Фрейнда. У животных ежедневно с 1 по 7 сутки отбирали по 500 мкл крови. Был выполнен мультиплексный цитокиновый тест с помощью набора реагентов Bio-Plex Pro Rat Cytokine 24-plex Assay на платформе Bio-Plex. Показано, что в контексте цитокинового профиля модель ЭАЭ у крыс отражает течение рассеянного склероза у человека в части динамики содержания системных лимфопролиферативных и гемо-поэтических факторов: IL-1b, IL-2, IL-4, IL-5, IL-6 и IL-7. В части динамики факторов таксиса лимфоцитов, моноцитов и других клеток иммунной системы изученная модель удовлетворительно имитирует динамику содержания IL-17, RANTES (CCL-5) и MCP-1 (CCL-2), но отличается по динамике GRO/KC (CXCL1). В отношении факторов, влияющих на цитотоксические и апоптотические реакции, сходство модели с заболеванием человека было выявлено по таким ключевым факторам, как IFNy, IL-6 и IL-17, но не по TNFa.
Ключевые слова: рассеянный склероз, экспериментальный аутоиммунный энцефаломиелит, миелин, иммунизация, мультиплексный цитокиновый тест
Благодарности: авторы благодарят Бориса Шевелева за участие в иммунизации животных.
Финансирование: исследование поддержано Министерством образования и науки Российской Федерации (Соглашение о предоставлении субсидии № 14.607.21.0133 от 27.10.2015, уникальный идентификатор RFMEFI60715X0133).
[X] Для корреспонденции: Удовиченко Игорь Петрович
Пр-т Науки, д. 6, г. Пущино, Московская обл., 142290; iudovichenko1@yandex.ru
Статья получена: 15.12.2017 Статья принята к печати: 20.12.2017
Multiple sclerosis (MP) is a sever neurodegenerative autoimmune disorder. Due to its high prevalence and the severity of symptoms causing partial or complete loss of mobility, multiple sclerosis remains a pressing problem, prompting a search for new therapies. Most patients with MS completely loose the mobility 25 years after the onset of the disease. More than a half of MS patients become dependent on crutches 15 years after appearance of the first symptoms. To date, there is no effective causal treatment for MS.
Usually the disease strikes at young age: 70 % to 80 % of patients suffer the first symptoms of MS between 20 and 40 years of age [1]. MS is diagnosed by neurological examinations, magnetic resonance imaging of the central nervous system, and by biopsy or autopsy [2]. MS has numerous clinical manifestations indicating damage to the spinal cord, the brain, cranial nerves, the cerebellum, and cognitive function. Current diagnostics are insufficient for accurate estimation of MS severity. MRI, electroencephalography and lumbar puncture can still be inconclusive, in spite of providing valuable information about patient's condition. In patients with MS, many symptoms can be caused by infection, vascular pathology, or autoimmune comorbidities [3].
There are four types of MS: relapsing-remitting (RRMS, alternating periods of relapses and remissions) occurring in 80 % to 85 % of patients; primary progressive (PPMS) occurring in 10 % to 15 % of patients; progressive-relapsing (PRMS) — in 5 % of patients; and secondary-progressive (SPMS) [4, 5]. About half of patients with RRMS develop symptoms of SPMS 10 years after the onset of the disease. Over 90 % of patients with RPMS eventually demonstrate SPMS symptoms [6].
The hallmark of MS is destruction of the myelin sheaths of neurons in the central nervous system caused by clustering T- and B-cells. Another typical feature of this disease is accumulation of oligoclonal antibodies in the cerebrospinal fluid. It is not clear, though, how and where the clonal expansion of lymphocytes specific for myelin basic protein is initially triggered. We do not know yet whether it happens in the CNS, where the myelin sheath is directly involved, or outside of it, with autoreactive species migrating to the CNS from other places [7].
Development of effective MS treatments is impossible without animal models accurately replicating the course of the disease in humans, such as experimental autoimmune encephalomyelitis (EAE) of rats and mice. EAE is induced by injecting myelin or basic myelin protein (MBP) suspensions in incomplete Freund's adjuvant into the hind footpads of rodents [8]. One month after immunization the mice develop hind limb paralysis which lasts for 4-6 months [9]. In Dark Agouti (DA) rats, EAE progresses more rapidly (paralysis sets in on days 10-11 and lasts until day 14). The key difference of EAE in animals from MS in humans is full recovery of rodents, which is absolutely unattainable for humans at this point.
An interesting study [10] reports cytokine profiles of 19 patients with MS, including 16 patients with RRMS, 1 individual with PPMS, and 2 — with SPMS. The patients were distributed into groups based on disease duration from the moment of diagnosis: 4.2 ± 0.8 months in group 1 and 76.6 ± 14.3 months in group 2. The study showed that in earlier stages of MS (in comparison with later stages and the absence of the disease), interferon gamma (IFNy) and the anti-inflammatory lymphokine IL-10 dominate in the cytokine profiles. In the late stage, the levels of IL-1RA, IL-8, IL-12(p70), CCL-3, CCL-7, CCL-11, CXCL-10, FGF, and IFNy go down. Later stages are also characterized by elevated levels of IL-1a, IL-1b, IL-2RA, IL-3, IL-4, IL-7, IL-12(p40), IL-18, CCL-5 (RANTES), CCL-27,
HGF, MIF, M-CSF and TRAIL. Interestingly, MS patients were shown to have elevated blood levels of IL-17, known to play a key role in triggering development of psoriatic skin lesions [11]. In addition, patients with RRMS exhibited elevated IL-22 levels. Dynamics of cytokine profiles in the cerebrospinal fluid drove the researchers [10] to the conclusion about the crucial role of the accumulating IFNy and MIF (a key factor of joint capsule degeneration in osteoarthritis) and a few other factors stimulating migration of lymphocytes: CCL-5 (RANTES), CCL-2 and CCL-27, induced by IFNy and MIF. The study also revealed accumulation of proapoptotic TNF-a and TRAIL-ligand in the cerebrospinal fluid (but not blood) of MS-stricken patients.
These data suggest a few patterns typical for MS, including increased long-term systemic activity of hematopoietic growth factors, in particular those targeting granulocytes, sustained Th1-response, and overrepresentation of lymphocyte/ monocyte migration factors in the absence of pronounced proinflammatory response (factors stimulating production and taxis of neutrophils). The study [10] could provide an insight into how cytokine levels observed in the cerebrospinal fluid and blood change in patients with MS, but due to the limitations of the applied statistical methods, significance of the identified patterns is questionable.
Considering the above said, our study aimed to
1) investigate the short-term dynamics of cytokines in rats with rapidly progressing induced EAE;
2) compare the data on cytokine levels in patients with MS and in rats with induced EAE in order to assess the feasibility of the EAE rat model for testing anti-MS candidate drugs.
METHODS
Induction of EAE in rats
Experiments involving laboratory animals were carried out in compliance with the "Regulations for the use of Experimental Animals" (Addendum to Order 755 of the Ministry of Health of the USSR dated August 12, 1977) and the principles of the Declaration of Helsinki (2013).
Homogenates of the spinal cord of random-bred rats were prepared as described in [12]. Further in vivo experiments were carried out in Dark Agouti rats weighing 220-250 g. The main group included 11 animals. On day 0 the animals were injected with the spinal cord homogenate mixed with incomplete Freund's adjuvant in the ratio of 1 : 1 into the hind footpads. The total volume of the injected mixture was 100 pl per paw. The controls (n = 7) received 100 pl of normal saline mixed with incomplete Freund's adjuvant in the ratio of 1 : 1. From day 1 through day 7, except for day 6, blood samples were collected from the tail vein (500 pl of blood daily) and immediately used for serum preparation. Briefly, blood was placed into Vacuette Z serum sepclot activator vacuum test tubes and centrifuged for 15-20 min at 2,500 rpm and +4 °C. The obtained serum (about 100 pl) was transferred to microcentrifuge tubes and frozen at -20 °C. The animals were weighted daily, and the severity of the disease was assessed using the following scale: 0 points — no symptoms, 1 point — decreased tail tone, 2 points — impaired righting reflex, 3 points — partial paralysis, 4 points — complete paralysis, 5 points— moribund or dead. In borderline cases, a lower index value was opted. Clear signs of EAE appeared in the controls starting from day 8 to day 14 of the experiment. On days 11-14 the disease reached its peak, which lasted for 2-3 days.
Multiplex cytokine assay
Serum samples were analyzed on the Bio-Plex platform (BioRad, USA) using the Bio-Plex Pro Rat Cytokine 24-plex Assay (Bio-Rad). This assay employs magnetic beads coated with monoclonal antibodies to rat cytokines. It was performed according to the manufacturer's recommendations and the protocol published in [13]. Serum was divided into 50 pl aliquots for the analysis. Mean fluorescence intensity of each sample was measured on Luminex 200 analyzer (Luminex Corporation, USA). Data were processed using MasterPlex CT and MasterPlex QT analysis software (Hitachi Solutions America, USA). For each analyte a calibration curve was constructed using 7 concentrations expressed as pg per 1 ml serum.
Statistical analysis
Two quartiles and median values of cytokine levels in each group were calculated daily for each cytokine. Then, significance of differences between the groups was tested using the nonparametric Mann-Whitney test and Statistica 8.0 for Windows. At p-value > 0.05 the differences were considered insignificant; we also used 3 significance thresholds: p < 0.05, p < 0.01, and p < 0.001.
RESULTS
The data on the short-term dynamics of cytokine levels in human and animal blood are still scarce. Multiplex assays are expensive, and daily blood tests in MS patients and lab animals can be technically challenging or raise ethical concerns. Data obtained from the controls in the course of our experiment demonstrate that although incomplete Freund's adjuvant injected into the footpads does not induce EAE, it still causes considerable fluctuations of cytokine levels in animals' blood, rendering less reliable the assessment of the impact of the spinal cord homogenate on the course of the disease. Therefore, special statistical methods are needed to analyze the dynamics of cytokine profiles.
All animals included in the main group developed paralysis of the hind legs. The rising phase of the disease was observed on days 11-13, while the decline — on days 12-17. By day 18 all animals had recovered from the paralysis. Blood was collected on days 1 through 7 in the absence of visible signs of EAE.
Tables 1 and 2 show that on day 1 of the experiment the levels of 13 of total 24 analytes (IL-1a, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12(p70), IL-17, IL-18, G-CSF, IFN-y, RANTES (CCL-5), and MCP-1 (CCL-2) ) were significantly higher (by up to 220 % for IL-4) in the main group than in the controls in terms of the second and third significance thresholds (Fig. 1). On day 2 no significant differences were observed for all studied cytokines. On day 3 differences were observed for IL-1b and VEGF (< 0.05), but on day 4 again no differences were found. On day 5 the main group demonstrated a considerable decrease in the levels of IL-1a, IL-1b, IL-13, and erythropoietin (Fig. 2). On day 7 the differences between the groups were observed for 14 of 24 studied cytokines. Those were practically the same cytokines that showed differences on day 1, although statistical significance was confirmed for IL-10 and erythropoietin GM-CSF only and was not confirmed for IL-12(p70) and G-CSF (Fig. 3) Of note, the levels of 13 of 14 cytokines in the main group were higher than in the controls. The only exception was GM-CSF that dropped from 8.17 pg/ml to 2.00 pg/ml.
DISCUSSION
A cytokine burst on day 1 of the experiment followed by a drop on day 2 should be interpreted as a manifestation of acute clonal nonspecific response to excess myelin outside the CNS. The response to the myelin manifested as simultaneous release of several lymphoproliferative factors is likely to be stimulated by hyperproduction of IL-1b originating from macrophages, dendritic cells and skin fibroblasts.
Increased cytokine synthesis on days 5 and 7 is, most probably, the result of the step-by-step accumulation of various clonal-specific lymphocytes, including those with autologous reactivity to myelin. Such longitude of the reaction is typical for the systemic clonal expansion of T-cells and eventually leads to visible physiological symptoms.
The most significant differences between the main and the control groups on day 7 were observed for the levels of IL-18 (2,475.85/4,182.05 pg/ml), RANTES (756.78/1,310.78 pg/ml), MCP 1 (CCL 2) (1,909.68/3,300.50 pg/ml) and IL-2 (743.52/1,091.57 pg/ml). Considering that IL-2 has been proved to induce production of other growth and hematopoietic factors [14], an assumption can be made that IL-2 triggers synthesis of such nonspecific immune factors as VEGF and erythropoietin, as well as IL-13, whose synthesis lagged in phase with respect to IL-2. Considering persistently high levels of IL-2 typical for patients with MS [10], this lymphokine seems to play a key role in the mass proliferation of lymphocytes
Table 1. Significance of differences between the main and the control groups of animals calculated by using the Mann-Whitney test with Yates's correction for continuity. Hypothesis tested: the absence of significant differences between the samples. The result is presented as Fisher's p with three significance thresholds: p > 0.05 — the difference is insignificant; 0.01 < p < 0.05 — the first significance threshold; 0.001 < p < 0.01 — the second significance threshold; p < 0.001 — the third significance threshold
Cytokine Days of the experiment
1 2 3 4 5 7
IL-1a 3 - - - 2 2
IL-1b - - 1 - 2 -
IL-2 2 - - - - 1
IL-4 3 - - - - 2
IL-5 2 - - - - 2
IL-6 3 - - - - 1
IL-7 2 - - - - 2
IL-10 - - - - - 2
IL-12 2 - - - - -
IL-13 - - - - 2 -
IL-17 3 - - - - 2
IL-18 3 - - - - 2
Erythropoietin EPO - - - - 1 2
G-CSF 3 - - - - -
GM-CSF - - - - - 3
GRO/KC - - - - - -
IFN-y 2 - - - - 1
M-CSF - - - - - -
MIP-3a - - - - - -
RANTES 2 - - - - 2
TNFa - - - - - -
VEGF - - 1 - - -
Leptin - - - - - -
MCP-1 2 - - - - 2
Table 2. Statistical analysis of changing cytokine levels in the main group of rats with induced autoimmune encephalomyelitis and the controls
Cytokine Day of the experiment
Parameter Day 1 Day 2 Day 3 Day 4 Day 5 Day 7
Controls Main group Controls Main group Controls Main group Controls Main group Controls Main group Controls Main group
IL-1a Mean 195 387 182 206 268 181 193 144 319 138 203 359
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 87 92 65 61 148 122 113 76 279 75 84 134
Q25 132 304 133 170 160 68 98 83 182 90 139 218
Median 188 347 175 204 218 200 195 155 184 121 184 411
Q75 288 487 226 264 396 237 274 210 396 187 269 462
IL-1b Mean 433 401 412 274 1033 493 469 265 819 250 319 571
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 386 382 244 184 427 362 432 157 861 164 180 288
Q25 251 236 211 148 701 134 119 101 279 151 180 300
Median 315 270 399 222 967 441 412 310 607 214 204 536
Q75 411 367 550 370 1 477 782 553 409 730 307 504 821
IL-2 Mean 356 607 366 436 367 500 472 414 579 373 744 1092
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 103 138 127 207 209 303 292 180 286 215 245 348
Q25 278 579 285 319 186 311 213 260 396 205 543 776
Median 374 597 345 331 332 432 308 362 557 284 657 1145
Q75 443 626 463 629 523 608 762 647 668 509 914 1368
IL-4 Mean 11 36 9 19 9 24 14 18 19 16 41 90
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 6 16 4 19 8 23 12 16 13 15 27 40
Q25 6 27 5 5 4 4 4 5 9 4 17 57
Median 11 30 8 8 6 17 8 12 15 14 33 76
Q75 17 38 12 37 10 32 29 34 33 23 64 128
IL-5 Mean 77 128 59 69 56 88 69 74 91 68 136 193
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 25 27 33 51 47 59 62 45 43 49 46 34
Q25 55 110 26 22 23 26 22 20 43 22 106 174
Median 78 123 63 54 30 83 23 67 88 83 130 193
Q75 97 147 78 121 113 133 144 106 118 106 176 212
IL-6 Mean 232 503 351 273 468 1595 515 540 600 316 668 1145
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 79 204 226 255 404 3960 572 743 284 269 292 496
Q25 172 379 169 63 170 96 76 85 414 46 456 640
Median 224 444 340 182 349 428 190 240 584 261 521 1240
Q75 287 463 540 470 758 635 1 132 559 713 556 877 1624
IL-7 Mean 103 254 68 123 70 178 111 114 125 106 228 612
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 50 96 62 128 83 169 129 115 77 98 146 236
Q25 58 181 14 16 12 13 11 16 50 10 119 428
Median 102 226 63 72 23 144 20 64 109 108 161 724
Q75 145 350 90 246 159 281 239 224 209 165 391 787
IL-10 Mean 149 240 105 180 121 215 163 157 208 132 403 634
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 70 80 53 131 93 200 129 99 70 106 160 226
Q25 91 208 61 57 56 63 56 58 144 44 254 420
Median 142 220 101 183 67 149 88 116 214 116 413 557
Q75 217 280 129 284 237 277 294 272 287 208 550 821
Продолжение табл. 2
IL-12 Mean 46 125 35 56 43 81 58 54 68 47 155 288
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 29 62 35 61 56 102 68 61 50 59 98 143
Q25 16 88 5 6 5 6 6 6 15 4 81 173
Median 49 99 32 23 10 53 12 29 59 26 125 277
Q75 74 133 46 123 98 118 135 70 104 81 212 425
IL-13 Mean 31 32 15 16 20 22 22 13 19 13 33 61
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 28 31 8 15 8 20 26 11 6 8 21 37
Q25 18 16 9 6 13 9 7 6 15 6 19 29
Median 21 20 14 9 20 13 13 9 18 11 23 44
Q75 28 37 22 20 23 28 26 15 22 17 58 95
IL-17 Mean 24 55 17 25 19 36 26 27 36 25 67 119
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 12 20 15 28 24 36 32 23 22 24 38 43
Q25 12 43 3 3 3 3 3 4 16 3 38 84
Median 24 49 16 15 4 34 5 21 35 28 62 108
Q75 37 56 24 44 50 52 52 42 58 45 102 168
IL-18 Mean 1110 2360 909 1480 976 2004 1351 1829 1628 1562 2476 4182
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 340 822 345 828 600 1513 895 1390 455 1557 774 1495
Q25 846 1671 623 707 591 582 615 779 1494 426 1663 3219
Median 1009 2167 877 1444 609 1677 1064 1157 1616 1152 2334 3808
Q75 1462 2766 1094 2243 1701 3435 2055 2367 1831 2632 3217 4100
Erythropoietin EPO Mean 202 263 186 242 197 310 238 246 281 175 342 745
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 68 60 77 141 62 240 71 95 86 91 117 345
Q25 154 235 127 110 158 127 173 153 209 107 249 504
Median 175 258 179 219 202 272 209 223 247 166 360 585
Q75 235 287 225 346 241 352 300 346 373 197 415 1060
G-CSF Mean 3 6 3 4 3 6 4 4 5 4 9 15
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 1 3 1 3 2 6 3 2 1 2 6 9
Q25 3 4 2 2 2 2 2 2 3 2 4 8
Median 3 6 3 3 2 4 2 3 4 3 6 12
Q75 4 7 3 6 5 8 6 5 6 5 14 24
GM-CSF Mean 5 7 5 5 6 9 8 5 6 4 8 2
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 3 3 3 3 4 10 6 2 4 2 2 1
Q25 2 6 3 2 3 4 4 3 2 2 7 1
Median 5 7 6 5 6 9 9 5 5 5 8 2
Q75 6 10 8 8 9 10 10 7 11 5 10 3
GRO/KC Mean 161 237 156 165 217 153 201 90 153 116 163 178
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 93 104 79 65 134 97 115 31 128 43 62 57
Q25 72 124 101 135 75 64 84 62 82 74 103 153
Median 142 267 140 157 239 133 198 96 90 119 155 170
Q75 270 331 191 189 341 272 299 116 324 145 184 222
IFNy Mean 36 79 30 42 32 63 61 45 53 40 107 215
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 14 46 13 40 23 77 62 41 19 35 45 122
Q25 27 52 20 14 17 14 17 15 36 12 65 87
Median 30 64 27 20 20 43 24 28 52 27 98 206
Q75 46 74 41 60 56 64 95 66 76 69 148 290
Продолжение табл. 2
M-CSF Mean 132 174 92 89 91 121 100 79 93 104 113 164
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 42 44 46 38 70 83 116 49 54 60 55 74
Q25 99 143 64 55 32 53 32 33 39 41 52 110
Median 124 159 80 88 71 106 40 69 69 105 141 149
Q75 175 205 122 111 174 183 164 121 147 164 165 234
MIP-3a Mean 70 88 55 49 45 88 69 64 76 45 88 128
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 19 38 44 40 44 95 80 59 45 40 36 44
Q25 52 47 19 13 12 21 14 13 25 11 65 107
Median 76 94 41 31 21 53 19 51 91 34 83 139
Q75 86 122 85 83 99 134 161 83 122 90 102 171
RANTES Mean 685 1070 342 470 295 696 364 505 574 456 757 1311
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 230 247 277 422 306 541 403 381 208 389 147 388
Q25 545 966 80 73 108 60 37 51 437 39 726 814
Median 665 1052 334 387 139 679 57 545 634 660 758 1495
Q75 805 1129 558 855 481 1212 821 777 687 735 846 1580
TNFa Mean 58 39 25 30 29 33 30 28 33 27 54 82
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 26 10 15 18 18 19 20 14 11 19 26 42
Q25 42 29 17 16 16 16 17 16 21 14 32 51
Median 57 39 19 21 19 27 23 25 36 24 57 72
Q75 72 49 26 47 46 44 49 34 39 29 82 106
VEGF Mean 130 87 88 55 128 73 107 46 133 65 92 110
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 110 90 57 79 73 80 78 24 149 49 44 50
Q25 41 52 42 20 53 37 54 32 33 29 57 57
Median 119 60 88 35 129 44 78 36 97 46 84 100
Q75 132 74 128 44 178 84 185 58 136 89 146 139
Leptin Mean 158 217 59 131 121 264 129 90 231 213 239 460
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 161 240 77 184 163 255 202 167 219 199 237 358
Q25 10 1 20 11 11 16 3 4 12 17 0 20
Median 150 137 24 27 21 312 21 20 246 286 324 620
Q75 301 437 83 273 304 482 375 30 447 419 404 745
MCP-1 Mean 1979 2662 2277 2317 2428 3097 2071 2468 2117 2254 1910 3300
N 6 10 8 12 6 11 7 11 7 11 7 11
SD 413 365 949 754 576 1120 830 1141 830 623 557 879
Q25 1592 2353 1606 1729 2130 2126 1501 1575 1651 1889 1340 2986
Median 2102 2606 2155 2063 2428 3108 1849 2192 2012 2448 1946 3411
Q75 2317 2864 2909 3047 2904 3762 2369 3021 2189 2750 2405 3846
outside the CNS. Increasing levels of lymphoproliferative and hematopoietic IL-4, IL-5, IL-6, Il-7, and IL-13 in the backdrop of decreased GM-CSF can be described as a cascade induced with IL-2 participation.
Unlike MS of humans, EAE in rats is not accompanied by production of proapoptotic TNF-a, regardless of the increased synthesis of its classic inducers IL-12, IL-18 and IFNy [14]. Therefore, elevated levels of TNF-a in patients with MS are rather a result and not the cause of myelin destruction. At the same time, TNF-a can contribute significantly to the damage of astrocytes and neurons in the late stages of MS.
According to the pattern described in [10], simultaneous increase and decrease of IFNy and RANTES (CCL-5),
respectively, in rats with EAE simulate similar processes occurring in humans with MS. The early stages of EAE in rats are not accompanied by an increase in GRO/KC (CXCL1) responsible for lymphocyte infiltration in the CNS, which renders the rat model different from MS in humans [10].
Both rats with EAE and humans with MS have hyperproduction of IL-17 which can contribute to the accumulation of specific lymphocytes in the CNS and activate their toxic function.
In spite of IL-1b hyperproduction, MS in humans shows no signs of neutrophil involvement in the pathology, which is also true for the factors regulating neutrophil taxis and activation. This pattern turned to be no different in the studied rat model.
300
250 -
200
150 -
100
50
Controls J Main group
CC_QC\I^U}COr--OC\ICOr--COO
o raLL CCCßCCLL C
cLOTOT^^OTcolUI/CD
î lllll|| || CDSŒTSSÎÉ1-
t CD CD 1 CC
Î
<B d
-1 5
î
Fig. 1. Changes in cytokine levels in the blood serum of rats with induced EAE in comparison with the controls 1 day after the injection. Cytokine levels in the controls were taken as 100 %. Significant differences are marked with arrows
120
100 -
80
60
40 -
20
43
31
64
87
75
85
53
70
64
68
112
96
69
81
62
70
76
76
59
80
84
49
106
92
I Controls I Main group
I I I I I I I I I I I I I I I I I I I I I I I
CC_QC\l^riOCOr--OC\IC0r--C0O1J-1J-O raLL CCCßCCLL C T-
T î î
CD CD CC
î î
H. Q-
<B d
Fig. 2. Changes in cytokine levels in the blood serum of rats with induced EAE in comparison with the controls 5 days after the injection. Cytokine levels in the controls were taken as 100 %. Significant differences are marked with arrows
0
0
The levels of M-CSF stimulating proliferation of neutrophil precursors did not change throughout the experiment. The same pattern was observed for MIP-3a (CCL20) that protects mucosa from bacterial infection and for leptin that raises body temperature in infected individuals.
Hyperproduction of IL-4 and IL-10 in rats with EAE in the background of elevated IL-5, IL-13, and GM-CSF should be considered a factor stimulating proliferation of B-cells. In theory, this set of cytokines can trigger synthesis of oligoclonal antibodies, but this effect has not yet been described in the literature.
Our experiment proves that proliferation of myelin-specific lymphocytes can be triggered outside the CNS. However, the
course of EAE in rats and the course of MP in humans differ considerably. We cannot rule out that the first event occurring at the onset of the disease is infiltration of the CNS by lymphocytes that do not undergo clonal expansion but do undergo further selection in the presence of excess myelin. Abnormal behavior of lymphocytes observed in the rat model can be a result of their primary clonal-nonspecific hyperproliferation triggered by systemic or local excess of lymphoproliferative factors or/and lymphotaxis factors originating in CNS. Another possibility is induction of abnormally rapid degradation of myelin in CNS leading to a massive release of degradation products into the systemic circulation. In this case the rat model seems to be quite adequate to the early stages of MS in humans.
300 -1
250 -
200 -
150 -
100 -
50 -
268
221
117 179
147
171
142
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186 185
157
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169
169
Controls
Main group
201
109
25
193
173
145 146
150
120
173
„ l l l l l l l l l l l l l l l l l l „ . . „ . . .
CS-QC\l^rLOCOr~OC\ICOr--COOL1-L1-0 raLL CCCßCCLL Ст-
î îîîîî.^ î T î
CD CD 1 CC
î î
<ч О î
Fig. 3. Changes In cytokine levels In the blood serum of rats with Induced EAE In comparison with the controls 7 days after the Injection. Cytokine levels In the controls were taken as 100 %. Significant differences are marked with arrows
0
CONSLUSIONS
Data on the dynamics of cytokine production in rats with EAE obtained with the multiplex cytokine assay suggest that the rat model adequately imitates the course of MS in humans with respect to the levels of systemic lymphoproliferative and hematopoietic factors IL-1b, IL-2, IL-4, IL-5, IL-6 and IL-7. With
respect to factors regulating taxis of lymphocytes, monocytes and other immune cells, the model fairly well imitates behavior of IL-17, RANTES (CCL-5) and MCP-1 (CCL-2), but exhibits a different dynamics for GRO/KC (CXCL1) levels. The model resembles the course of MS in humans in terms of IFNy, IL-6 and IL-17 involved in cytotoxic and apoptotic reactions, but exhibits a different dynamics for TNF-a.
References
1. Noonan CW, Kathman SJ, White MC. Prevalence estimates for MS in the United States and evidence of an increasing trend for women. Neurology. 2002 Jan 8; 58 (1): 136-8.
2. Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol. 2000 Jun; 47 (6): 707-17.
3. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, Lublin FD et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol. 2001 Jul; 50 (1): 121-7.
4. Kremenchutzky M, Cottrell D, Rice G, Hader W, Baskerville J, Koopman W et al. The natural history of multiple sclerosis: a geographically based study. 7. Progressive-relapsing and relapsing-progressive multiple sclerosis: are-evaluation. Brain. 1999 Oct; 122 (Pt 10): 1941-50.
5. Confavreux C, Vukusic S, Moreau T, Adeleine P. Relapses and progression of disability in multiple sclerosis. N Engl J Med. 2000 Nov 16; 343 (20): 1430-8. DOI: 10.1056/ NEJM200011163432001.
6. Weinshenker BG, Bass B, Rice GP, Noseworthy J, Carriere W, Baskerville J et al. The natural history of multiple sclerosis: a geographically based study. I. Clinical course and disability. Brain. 1989 Feb; 112 (Pt 1): 133-46.
7. Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M et al. The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain. 2009 May; 132 (Pt 5): 1175-89. DOI: 10.1093/brain/ awp070.
8. Mensah-Brown EP, Shahin A, Garey LJ, Lukic ML. Neuroglial response after induction of experimental allergic encephalomyelitis insusceptible and resistant rat strains. Cell Immunol. 2005 Feb; 233 (2): 140-7. DOI: 10.1016/j.cellimm.2005.04.023.
9. Contarini G, Giusti P, Skaper SD. Active Induction of Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice. Methods Mol Biol. 2018; 1727: 353-60. DOI: 10.1007/978-1-4939-7571-6_26.
10. Khaibullin T, Ivanova V, Martynova E, Cherepnev G, Khabirov F, Granatov E et al. Elevated Levels of Proinflammatory Cytokines in Cerebrospinal Fluid of Multiple Sclerosis Patients. Front Immunol. 2017 May 18; 8: 531. DOI: 10.3389/fimmu.2017.00531.
11. Albanesi C, Scarponi C, Cavani A, Federici M, Nasorri F, Girolomoni G. Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes. J Invest Dermatol. 2000; 115(1): 81-7.
12. Beeton C, Garcia A, Chandy KG. Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis. J Vis Exp. 2007; (5): 224. DOI: 10.3791/224.
13. Poveschenko AF, Kazakov OV, Orlov NB, Poveschenko OV, Kim II, Bondarenko NA et al. Serum cytokines of Wistar rats — markers of carcinogenesisand effectiveness of cancer therapy. Fundamental research. 2015; 1 (Pt 8): 1664-70. Russian.
14. Hamblin AS. Lymphokines and interleukins. Immunology. 1988; 64 (Suppl 1): 39-41.
Литература
1. Noonan CW, Kathman SJ, White MC. Prevalence estimates for 8. MS in the United States and evidence of an increasing trend for women. Neurology. 2002 Jan 8; 58 (1): 136-8.
2. Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: 9. implications for the pathogenesis of demyelination. Ann Neurol. 2000 Jun; 47 (6): 707-17.
3. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, Lublin FD et al. Recommended diagnostic criteria for multiple 10. sclerosis: guidelines from the International Panel on the diagnosis
of multiple sclerosis. Ann Neurol. 2001 Jul; 50 (1): 121-7.
4. Kremenchutzky M, Cottrell D, Rice G, Hader W, Baskerville J, Koopman W et al. The natural history of multiple sclerosis: 11. a geographically based study. 7. Progressive-relapsing and relapsing-progressive multiple sclerosis: are-evaluation. Brain. 1999 Oct; 122 (Pt 10): 1941-50.
5. Confavreux C, Vukusic S, Moreau T, Adeleine P. Relapses
and progression of disability in multiple sclerosis. N Engl 12. J Med. 2000 Nov 16; 343 (20): 1430-8. DOI: 10.1056/ NEJM200011163432001.
6. Weinshenker BG, Bass B, Rice GP, Noseworthy J, Carriere W, 13. Baskerville J et al. The natural history of multiple sclerosis: a geographically based study. I. Clinical course and disability. Brain. 1989 Feb; 112 (Pt 1): 133-46.
7. Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M et al. The relation between inflammation and neurodegeneration in multiple sclerosis brains. 14. Brain. 2009 May; 132 (Pt 5): 1175-89. DOI: 10.1093/brain/ awp070.
Mensah-Brown EP, Shahin A, Garey LJ, Lukic ML. Neuroglial response after induction of experimental allergic encephalomyelitis insusceptible and resistant rat strains. Cell Immunol. 2005 Feb; 233 (2): 140-7. DOI: 10.1016/j.cellimm.2005.04.023. Contarini G, Giusti P, Skaper SD. Active Induction of Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice. Methods Mol Biol. 2018; 1727: 353-60. DOI: 10.1007/978-1-4939-7571-6_26.
Khaibullin T, Ivanova V, Martynova E, Cherepnev G, Khabirov F, Granatov E et al. Elevated Levels of Proinflammatory Cytokines in Cerebrospinal Fluid of Multiple Sclerosis Patients. Front Immunol. 2017 May 18; 8: 531. DOI: 10.3389/fimmu.2017.00531. Albanesi C, Scarponi C, Cavani A, Federici M, Nasorri F, Girolomoni G. Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes. J Invest Dermatol. 2000; 115(1): 81-7.
Beeton C, Garcia A, Chandy KG. Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis. J Vis Exp. 2007; (5): 224. DOI: 10.3791/224. Повещенко А. Ф., Казаков О. В., Орлов Н. Б., Повещен-ко О. В., Ким И. И., Бондаренко Н. А. и др. Цитокины сыворотки крови как маркеры онкогенеза и эффективности терапии при экспериментальной опухоли молочной железы крыс Wistar. Фундаментальные исследования. 2015. 1 (ч. 8): 1664-70.
Hamblin AS. Lymphokines and interleukins. Immunology. 1988; 64 (Suppl 1): 39-41.
