Increased interleukin-10 associated with low IL-6 concentration correlated with greater survival...

Increased interleukin-10 associated with low IL-6

concentration correlated with greater survival rates

in mice infected by rabies virus vaccinated against

it and immunomodulated with P. acnes

J. Megida,*, R. Kanenob, C.N. Nozakia, C.J.C. Britoa, M.F. Almeidac

aFaculdade de Medicina Veterinaria, Department of Veterinary Hygiene and Public Health,

UNESP, Botucatu SP, BrazilbInstituto de Ciencias Biologicas, Department of Microbiology and Immunology, UNESP, Botucatu, SP, Brazil

cCentro de Controle de Zoonoses, Sao Paulo, SP, Brazil

Accepted 19 December 2003

Abstract

Macrophage activity, cytokines serum concentration, serum neutralizing antibodies and lethality

by rabies were evaluated in swiss mice experimentally infected with street rabies virus and

submitted or not to antirabies vaccination and immunomodulation with P. acnes. Animals were

killed at different times and serum was collected in order to evaluate cytokines concentration;

peritonial and splenic macrophages were collected for macrophage activity evaluation. Greater

survival rates higher IL-10 and low IL-6 serum concentration were observed in vaccinated animals

treated using P. acnes.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: Rabies; Cytokines; Immunomodulation; Vaccine; Macrophage; IL-10; IL-6

Resume

L’activite des macrophages, la concentration serique des cytokines, le taux d’anticorps

neutralisants et le taux de mortalite ont ete evalues chez des souris infectees par le virus rabique

des rues et soumises ou non a une vaccination antirabique et a une immunomodulation avec P. acnes.

Les animaux ont ete sacrifies a des temps differents et le serum a ete collecte afin d’evaluer la

concentration en cytokines.

Les macrophages peritoneaux et spleniques ont ete collectes afin d’evaluer l’activite

macrophagique.

0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cimid.2004.01.002

Comparative Immunology, Microbiology

& Infectious Diseases 27 (2004) 393–411

www.elsevier.com/locate/cimid

* Corresponding author.

E-mail address: [email protected] (J. Megid).

http://www.elsevier.com/locate/cimid

Un taux de survie plus eleve et un taux de concentration plus eleve en IL10 et moins eleve en IL6

ont ete observes chez les animaux vaccines et traites avec P acnes.

q 2004 Elsevier Ltd. All rights reserved.

Mots-cle: Rage; Cytokine; Immunomodulation; Vaccin; Macrophage; IL-10; IL-6

1. Introduction

Several researchers [1–4] have studied the role of macrophages in rabies in relation to

virus sequestration, greater activity in susceptible animals and persistence of rabies virus

inside them [4].

The possible role of cytokines in rabies pathogenesis in relation to the concentration of

IL-1a, IL-1b and TNF-a and their brain receptors has been evaluated by other researchers

[5–7].

The use of immunomodulators, antiviral drugs and interferon in rabies was reported by

some authors [8–11]. The mechanism of immunomodulating using Propionibacterium

acnes (P. acnes) has been studied by several researchers [12–14] both in human and

animal models of infectious diseases [15,16].

P. acnes action was evaluated in mice infected by rabies virus and researchers

observed greater survival rates in animals treated with the immunomodulator and

antirabies vaccine. This greater survival was not related to serum neutralizing antibodies,

interferon gamma concentration and cellular immunity evaluated by the Macrophage

Inhibition Test [17] but it was correlated with higher Natural Killer activity in infected

mice treated using P. acnes [18].

Considering the action of P. acnes in macrophages activity and its possible role in the

protection against rabies, the aim of the present trial was to evaluate macrophage activity

and cytokine concentration in mice infected by rabies virus, vaccinated against it and

submitted to treatment with P. acnes (Corynebacterium parvum).

2. Material and methods

2.1. Animals

Outbred, female, swiss albino mice, approximately four weeks old, from the Central

Laboratory Animal Facility at UNESP-Botucatu.

2.2. Rabies virus

Virus sample used was isolated from the brain of a rabid dog positive for rabies both by

the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The virus

sample was maintained using one intracerebral and one IM passage in mice and stored at

220 8C. Virus titration was accomplished by 0.03 ml IM inoculation of serial 10-fold viral

dilutions in diluent using 10 mice per dilution. Viral titer was 1021.29 DL50/0.03 ml IM.

J. Megid et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 393–411394

2.3. Immunomodulator

P. acnes produced by Laboratorio Farmaceutico do Estado de Pernambuco (LAFEPE),

constituted of 4 mg/2 ml of P. acnes (formerly C. parvum), dose used was 0.2 ml (0.4 mg)

per animal.

2.4. Vaccine

Fuenzalida-Palacios anti-rabies vaccine produced by TECPAR (Instituto de Tecnologia

do Parana), dose used was 40 ml in the left foot pad.

2.5. Animal inoculation

Performed according to the methodology described by Koprowsky [19], with small

modification related to the route of inoculation (IM route into the right hind limb).

2.6. Evaluation of macrophage activity

Hydrogen peroxide was evaluated through the technique by Reed et al. [20] and nitrite

production was performed according to Green et al. [21]. Results, expressed in mM, were

evaluated in splenic and peritonial macrophages non-stimulated or stimulated using

Interferon-g (IFNg-).

2.7. Serum neutralization (SN)

The technique adopted was described by Favoretto et al. [22], using in PV strain and

BHK21 cell culture; sera were first diluted 1:5 and then used in two-fold serial dilutions up

to 1:40. SN titers were expressed in IU/ml.

2.8. Cytokine assay

Tumour Necrosis Factor-a (TNF-a), interferon-g (IFN-g), Interleukin 1a (IL1a), Inter-

leukin1b (IL1b), Interleukin-2 (IL-2), Interleukin 3 (IL-3), Interleukin 4 (IL-4),

Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 10 (IL-10) and Interleukin 12 (IL-12)

were evaluated through ELISA test, using pooled sera from three mice for each colletion

time, according the methodology recommended by the manufacturer (R and D Systems).

2.9. Statistical analysis

Performed by Kruskall–Wallis [23].

J. Megid et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 393–411 395

3. Experimental procedures

This trial was conducted using six experimental groups with 300 mice in each; animals

were submitted to the following treatments.

Infected group. Intramuscularly (IM) injected using 0.03 ml of rabid mouse brain

suspension.

Infected P. acnes group. IM inoculation with 0.03 ml of rabid mouse brain suspension

and treated 24 h after inoculation with 0.2 ml P. acnes suspension by (IP) route.

Infected vaccinated P. acnes: group. IM inoculation with 0.03 ml of rabid mouse brain

suspension and 24 h after that, vaccinated using 0.04 ml Fuenzalida-Palacios anti-rabies

vaccine IM and treated with 0.2 ml P. acnes IP.

Vaccinated: group: IM inoculation with 0.03 ml of normal mouse brain suspension and

vaccinated using 0.04 ml Fuenzalida-Palacios anti-rabies vaccine IM.

Control group. 0.03 ml IM injection of normal mouse brain suspension (NBS).

P. acnes control group. 0.03 ml IM injection of NBS and treated with P. acnes

suspension by IP route, 24 h after inoculation.

After immunomodulator treatment (24 h after infection) 15 mice were killed after 24 h,

7,14 and 21 d. A pool of serum from three mice colleted at these different times was

promptly submitted to cytokine assay. For the macrophage activity evaluation peritonial

macrophagos were obtained by perfusion and spleen samples were colleted for adherent

cells at the same time.

4. Results

Greater survival rate were observed in the group treated using P. acnes and anti-rabies

vaccine (50%) followed by the group only treated with P. acnes (29.2%) and infected

animals (25%). Survival rate are presented in Fig. 1.

The highest titers of serum neutralizing antibodies, correlated with clinical symptoms

in the animals, were detected after 14th day in all infected groups (Table 1; Fig. 2) Results

were not statistically significant ðp . 0:05Þ:

Production of nitrite was not detected in spleen macrophages of any groups. Higher

production was observed in IFNg stimulated peritonial macrophages of the P. acnes

Fig. 1. Survival rate of infected mice submitted to different treatments.


control group on the 7th day compared to control group ðp , 0:01Þ (Table 2).

No difference was observed in non-stimulated peritonial macrophages ðp . 0:05Þ

(Table 3).

Hydrogen peroxide production of non-stimulated and IFNg stimulated peritonial

macrophage was similar in all moments and groups ðp . 0:05Þ (Tables 4 and 5).

In non-stimulated spleen macrophages, higher peroxide production was observed in the

P. acnes control group, compared to control group at 24 h ðp , 0:05Þ: Inversely, the P:acnes

control group ðp , 0:01Þ and the vaccinated group ðp , 0:05Þ presented the lowest

macrophage activity on the 7th day when compared to the infected groups submitted or not

to immunomodulation and vaccination. On the 14th day, greater activity was verified in the

vaccinated group ðp , 0:05Þ and the lowest activity in P. acnes control group ðp , 0:001Þ:

No difference was verified on the 21st day ðp . 0:05Þ:

When stimulated using IFN-g, the greatest activity was observed in the P. acnes control

group, in the infected, and infected P. acnes groups when compared to the control group

ðp , 0:05Þ at 24 h. On the 7th day the highest peroxide production was detected in all

infected groups (submitted or not to vaccination and immunomodulation) compared to

non-infected ones ðp , 0:001–0:05Þ: Highest activity was detected on the 14th day in

the group only vaccinated compared to the control ones ðp , 0:01Þ and compared to

the infected, vaccinated group ðp , 0:05Þ: No difference was verified on the 21st day

ðp . 0:05Þ: Results are presented in Tables 4 and 5.

Table 1

Serum Neutralizing antibodies in infected mice submitted to different treatments. Results expressed as median

(mean ^ standard deviation) of IU

24 h 7 d 14 d 21 d

Infected 0.18(0.256 ^ 0.170) 0.56(0.904 ^ 0.831) 1.04(0.806 ^ 0.323) 0.78(0.747 ^ 0.360)

Infected/P. acnes 0.23(0.23 ^ 0.094) 0.203(0.226 ^ 0.095) 0.813(0.906 ^ 0.382) 0.64(0.64 ^ 0.08)

Infected vaccine/

P. acnes/

0.14(0.185 ^ 0.085) 0.36(0.585 ^ 0.435) 1.04(0.981 ^ 0.244) 0.91(0.857 ^ 0.204)

Fig. 2. Serum neutralizing antibody titers expressed in IU/ml, in mice infected by rabies and submitted or not to

anti-rabies vaccination and P. acnes treatment (median).


Table 2

Activity of interferon stimulated macrophages evaluated by nitrite production in the different groups and moments. Results expressed as median (mean þ standard

deviation)

Macrophages Day Control Control P. Acnes Infected Infected P. acnes Infected

Vaccinated

P. acnes

Vaccinated

Peritoneal 24 h 0 (42.369 þ 88.099) 0 (0 þ 0) 0 (0.009 þ 0.0145) 0 (10.203 þ 27.886) 0 (0.167 þ 0.047) 0 (29.781 þ 78.133)

7 d 0 (12.675 þ 43.910) 163.039 (180.154 þ 158.504) 0 (109.439 þ 295.486) 0 (54.041 þ 116.058) 0 (37.321 þ 58.163) 3.193 (29.368 þ 55.394)

14 d 0 (38.023 þ 60.354) 0 (50.108 þ 82.107) 0 (8.583 þ 18.335) 0 (33.411 þ 74.709) 0 (36.221 þ 78.836) 0 (29.396 þ 51.588)

21 d 0 (31.602 þ 71.809) 0 (65.548 þ 126.600) 0 (15.857 þ 38.931) 0 (69.069 þ 89.922) 0 (27.487 þ 48.627) 0 (12.597 þ 25.360)

Spleen 24 h 0 (0 þ 0) 0 (0 þ 0) 0 (0.002 þ 0.006) 0 (0.005 þ 0.0101) 0 (0.008 þ 0.013) 0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 0 (4.551 þ 20.354) 0 (3.683 þ 11.347) 0 (0 þ 0) 0 (0 þ 0)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

21 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

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Table 3

Activity of non-stimulated macrophages evaluated by nitrite production in the different groups and moments. Results expressed as median (mean þ standard deviation)

Macrophages Day Control Control P. acnes Infected Infected P. acnes Infected

vaccinated

P. acnes

Vaccinated

Peritoneal 24 h 0 (29.80 þ 77.800) 0 (0 þ 0) 0 (0.101 þ 0.015833) 0 (2.926 þ 11.667) 0 (0.007 þ 0.014) 0 (5.170 þ 11.856)

7 d 0 (11.637 þ 40. 313) 0 (66.223 þ 119.576) 0 (26.231 þ 81.793) 0 (42.661 þ 88.255) 0 (30.735 þ 55.358) 0 (23.133 þ 53.098)

14 d 0 (16.262 þ 36.298) 0 (43.641 þ 83.788) 0 (6.028 þ 13.479) 0 (29.078 þ 65.020) 0 (23.465 þ 62.082) 2.371 (50.402 þ 69.119)

21 d 0 (13.218 þ 45.127) 0 (61.875 þ 131.574) 0 (14.396 þ 36.192) 30.209 (73.203 þ 95.553) 0 (24.921 þ 55.210) 0 (5.601 þ 17.712)

Spleen 24 h 0 (0 þ 0) 0 (0 þ 0) 0 (0.002 þ 0.004) 0 (0.005 þ 0.0120) 0 (0.009 þ 0.015) 0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 0 (2.419 þ 6.344) 0 (2.582 þ 11.548) 0 (0 þ 0) 0 (0 þ 0)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

21 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

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Table 4

Activity of non-stimulated macrophages evaluated by hydrogen peroxide production in the different groups and moments. Results expressed as median

(mean þ standard deviation)

Macrophages Day Control Control P. acnes Infected Infected P. acnes Infected Vaccinated

P. acnes

Vaccinated

Peritonial 24 h 0 (0 þ 0) 0 (0.008 þ 0.020) 0.001 (0.141 þ 0.253) 0 (0.065 þ 0.155) 0 (5.170 þ 11.856)

7 d 0.078 (0.088

þ 0.0844)

0.004 (0.007 þ 0.012) 0.05 (0.348 þ 0.545) 0.003 (0.270 þ 0.582) 0 (0.311 þ 0.372) 0 (23.133 þ 53.098)

14 d 0.046 (0.085

þ 0.092)

0.015 (0.155 þ 0.058) 0.027 (2.474 þ 6.441) 0 (1.165 þ 2.787) 0.031 (2.655 þ 5.903) 2.372 (50.402

þ 69.119)

21 d 0 (0 þ 0) 0 (0.0004 þ 0.0012) 0 (0.024 þ 0.072) 0 (0.040 þ 0.060) 0 (0,040 þ 0.110) 0 (5,601 þ 17,712)

Spleen 24 h 0 (0 þ 0) 0.111 (0.116 þ 0.039) 0 (0.040 þ 0.093) 0.0003 (0.0971

þ 0.1675)

0.0003 (0.099 þ 0.135) 0 (0.463 þ 0.984)

7 d 0.003 (0.047

þ 0.070)

0.00004 (0.002

þ 0.002)

0.1695 (0.239

þ 0.293)

0.066 (0.231 þ 0.386) 0.0275 (0.259 þ 0.406) 0 (0.040 þ 0.119)

14 d 0 (0.030 þ 0.042) 0 (0 þ 0.0121) 0 (1.928 þ 3.814) 0 (1.827 þ 4.484) 0,001 (1.845 þ 4,322) 0,233 (0.235

þ 0.045)

21 d 0 (0.040 þ 0.080) 0 (0.002 þ 0.009) 0.0007 (0.0401

þ 0.0619)

0.015 (0.042 þ 0.061) 0 (0.002 þ 0.008) 0 (0.004 þ 0.008)

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Table 5

Activity of interferon stimulated macrophages evaluated by hydrogen peroxide production in the different groups and moments. Results expressed as median

(mean þ standard deviation)

Macrophages Day Control Control P. acnes Infected Infected P. acnes Infected Vaccinated

P. acnes

Vaccinated

Peritoneal 24 h 0 (0 þ 0) 0.004 (0.021 þ 0.030) 0.0005 (0.2192

þ 0.4238)

0 (0.381 þ 0.995) 0 (0.154 þ 0.335) 0 (29.781 þ 78.133)

7 d 0.069 (0.077

þ 0.080)

0.004 (0.004 þ 0.004) 0.064 (0.313 þ 0.404) 0 (0.297 þ 0,345) 0.006 (0.253 þ 0.384) 3.193(29.368 þ 55.394)

14 d 0.201 (0.211

þ 0.188)

0 (0.164 þ 0.116) 0.146 (1.348 þ 3.272) 0.023 (0.582 þ 1.195) 0.001 (3.911 þ 6.659) 0 (29.396 þ 51.588)

21 d 0 (0 þ 0) 0 (0.001 þ 0.003) 0 (0.039 þ 0.0601) 0 (0.062 þ 0.085) 0 (0.035 þ 0.073) 0 (12.597 þ 25.360)

Spleen 24 h 0 (0 þ 0) 0.083 (0.117 þ 0.093) 0.124 (0.242 þ 0.355) 0.072 (0.210 þ 0.263) 0.002 (0.152 þ 0.263) 0.048 (0.803 þ 2.32)

7 d 0.003 (0.057

þ 0.100)

0.002(0.0067 þ 0.010) 0.091 (0.282 þ 0.333) 0.067 (0.310 þ 0.489) 0.04 (0.349 þ 0.691) 0 (0.0006 þ 0.0018)

14 d 0 (0.057

þ 0.076)

0.016 (0.016 þ 0.049) 0.092 (0.732 þ 2.127) 0.036 (0.807 þ 2.546) 0.001 (1.730 þ 4.618) 0.21725 (0.218 þ 0.072)

21 d 0 (0.012967

þ 0.048317)

0.002 (0.086 þ 0.219) 0.003 (0.061 þ 0.080) 0.048 (0.064 þ 0.098) 0 (0.012 þ 0.030) 0 (0.005 þ 0.010)

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Table 6

Results of IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-10 and TNF-a in the different groups and moments. Values expressed as median (mean and standard deviation) pg/ml,

Botucatu-SP, 2000

Test Day Control Control P. acnes Infected Infected P. acnes Infected

vaccinated

P. acnes

Vaccinated

IL-1b 24 h 0 (7.584 þ 9.572) 5.19 (6.326

þ 5.799)

17.7 (9.862

þ 14.442)

4.79 (19.479

þ 22.018)

3.84 (13.737

þ 18.226)

25.164 (29.462

þ 9.304)

7 d 2.8 (5.170 þ 6.303) 4.51 (13.218

þ 24.859)

1.53 (4.889

þ 7.696)

29.58 (16.518

þ 24.418)

32.92 (6.428

þ 9.113)

6.444 (8.662

þ 5.739)

14 d 0 (6.754 þ 10.572) 202.55 (202.557

þ 247.152)

2.55 (3.210

þ 5.749)

0 (7.432

þ 9.022)

0 (2.902

þ 4.044)

35.147 (32.928

þ 6.528)

21 d 8.79 (7.614

þ 7.369)

1.78 (122.067

þ 262.885)

8.58(4.523

þ 5.580)

6.98 (10.161

þ 10.870)

2.44 (4.767

þ 5.198)

16.428 (19.617

þ 10.158)

IL-2 24 h 0 (3.927

þ 6.580)

0 (1.202

þ 3.602)

0 (1.314

þ 3.488)

0 (1.524

þ 3.139)

7.29 (9.614

þ 6.875)

0 (0 þ 0)

7 d 0 (7.353

þ 13.847)

0 (5.227

þ 12.909)

0 (7.602

þ 23.904)

0 (1.927

þ 3.165)

43.55 (5.008

þ 3.405)

0 (0 þ 0)

14 d 37.23 (37.915

þ 36.720)

0.88 (0.884

þ 1.250)

0 (2.516

þ 5.036)

0 (0 þ 0) 0 (3.148

þ 5.329)

0 (0 þ 0)

21 d 1.58(2.527

þ 2.941)

0 (8.107

þ 9.929)

3.32 (9.580

þ 11.599)

6.55 (0.022

þ 0.055)

5.44 (6.803

þ 7.565)

5.262 (8.845

þ 11.079)

IL-4 24 h 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0.971

þ 1.681)

21 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 1.372 (3.770

þ 5.070)

IL-5 24 h 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 0 (0.907

þ 2.487)

0 (0 þ 0) 0 (0.718

þ 2.155)

0 (0 þ 0)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0.144 þ 0.479) 0 (0 þ 0) 0 (0.895

þ 1.822)

0 (1.595

þ 2.763)

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21 d 0 (0 þ 0) 0 (0 þ 0) 0 (15.422

þ 32.283)

0 (0 þ 0) 0 (0.801

þ 1.316)

0 (12.346

þ 21.385)

IL-6 24 h 0 (102.880

þ 394.541)

0 (115.005

þ 201.726)

111.62 (150.503

þ 146.124)

135.44 (243.794

þ 250.935)

0 (203.294

þ 424.354)

283.95 (256.413

þ 118.342)

7 d 0 (0 þ 0) 0 (39.659

þ 99.589)

11.87 (85.518

þ 185.202)

6.66 (27.632

þ 54.595)

0 (0.536

þ 1.932)

112.74 (161.763

þ 191.051)

14 d 0 (22.568

þ 38.048)

1.47 (1.473

þ 2.083)

76.64 (104.757

þ 158.328)

0 (290.072

þ 540.049)

0 (32.275

þ 76.991)

110.74 (224.877

þ 232.565)

21 d 0 (0 þ 0) 8.093 (8.093

þ 26.841)

9.64 (43.995

þ 102.543)

0 (22.432

þ 30.121)

0 (16.429

þ 32.645)

332.58 (374.103

þ 281.769)

IL-10 24 h 0 (1.820

þ 7.051)

0 (5.238

þ 11.396)

0 (0.556

þ 1.478)

0 (3.267

þ 7.715)

56.97 (44.023

þ 40.904)

0 (0.715

þ 1.238)

7 d 0 (0.465 þ 0.928) 0 (1.091

þ 2.342)

0 (2.288

þ 6.287)

0 (2.808

þ 5.720)

0 (27.097

þ 27.593)

108.7 (107.299

þ 42.778)

14 d 7.12 (27.539

þ 39.819)

0 (0 þ 0) 0 (2.672

þ 4.871)

0 (0 þ 0) 0 (83.829

þ 106.840)

118.52 (105.432

þ 23.890)

21 d 0 (7.484

þ 20.370)

0 (8.395

þ 26.028)

0 (3.759

þ 10.239)

0 (0 þ 0) 25.80 (48.630

þ 47.521)

142.35 (141.883

þ 6.322)

TNF-a 24 h 0 (2418.6

þ 9367.1)

8 (10.505

þ 25182.9)

0 (5022.154

þ 12643.37)

0 (7297.8

þ 14653.79)

0 (24.151

þ 63.898)

0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 22 (276

þ 1203.056)

0 (4169.382

þ 8690.956)

0 (2835.231

þ 7872.247)

0 (29.941

þ 51.859)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (62.295

þ 196.994)

0 (0 þ 0)

21 d 0 (0 þ 0) 0 (0.086 þ 0) 0 (0 þ 0) 0 (42.965

þ 67.807)

0 (0 þ 0) 18.424 (53.438

þ 77.153)

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IFN-g, IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-10 and TNF-a were evaluated in all groups.

IL-1a, IL-3 and IL-12 were not evaluated in the vaccinated group.

IFN-g was not detected in any moment and group (results not shown).

Compared to all the other groups, the infected, vaccinated group presented higher IL-2

concentration at 24 h (Table 6 and Fig. 3); higher IL-10 concentration in all moments

Fig. 3. IL2 concentration (pg/ml) of different groups and at different moments. Values expressed in Mean of Ranks.

Fig. 4. IL-10 serum concentration (pg/ml) of different groups and at different moments. Values expressed in Mean

of Ranks.


except in relation to the vaccinated group (Table 6 and Fig. 4). Lower IL-6 concentrations

on the 7th and 21st day were observed when compared to the infected and to vaccinated

group, respectively (Table 6 and Fig. 5).

The vaccinated group presented IL-10 concentration similar to that of the infected,

vaccinated group in all moments except at 24hs (Table 6; Fig. 4); higher IL-6 (Table 6;

Fig. 5) and IL-4 (Table 6; Fig. 6) on the 21st day were observed, when compared to all the

other groups, except the infected P. acnes group. Higher levels of TNF-a on the 21st day

(Table 6; Fig. 7) were observed, when compared to all groups.

The concentration of IL-6 at 24 h in infected group was similar to the infected P. acnes

group at this moment, and both concentrations were higher than that of the control group.

Higher IL-6 concentration on the 7th day was observed in the infected group compared to

the infected, vaccinated group (Table 6; Fig. 5). Lower IL-10 concentration in all

moments, except on the 14th day, was observed in this group, compared to the infected

vaccinated and the vaccinated group (Table.6; Fig.4. IL-3 was greater in the infected group

on the 14th day compared to the other group (Table 7).

Fig. 5. IL-6 serum concentration of different groups and at different moments. Values expressed in Mean of Ranks.

Fig. 6. IL-4 serum concentration of different groups and at different moments. Values expressed in Mean of Ranks.


The infected P. acnes group presented higher IL-6 concentration at 24 h compared to

the control group, and similar IL-6 concentration compared to the vaccinated group on the

21st day (Table 6; Fig. 5). Lower IL-10 concentration in all moments (Table 6; Fig. 4) was

observed when compared to the infected vaccinated and the only vaccinated groups.

Higher concentration of TNF-a was observed in this group on the 14th day in relation to

all groups except the vaccinated one (Table 6; Fig. 7).

Higher IL-1a at 24 h was observed in all infected groups submitted or not to P:acnes

treatment and anti-rabies vaccine. The infected vaccinated P. acnes group presented the

highest concentration of this cytokine on the 14th day (Table 7; Fig. 8).

IL-1b (Table 6), IL-5 (Table 6) and IL-12 (Table 7) concentration was similar in all

moments and for all groups.

5. Discussion

A greater survival rate was observed in the animals treated using P. acnes and anti-

rabies vaccine, with not relation with the levels of serum neutralizing antibodies, what is in

agreement with the reports of several researchers [24,25]. In previous trials, survival rates

of mice infected by rabies virus, submitted to anti-rabies vaccine, and treated with P. acnes

or anti-rabies vaccine associated with P. acnes as post-exposure treatment, were studied,

and similar results were obtained in this experiment; the best survival rate was observed in

the vaccinated P. acnes treated group, followed by the P. acnes immunomodulated group;

the vaccinated group presented the lowest survival rate [17].

Higher levels of IL-10 in all moments except 24 h, IL-6 on the 7th day, and IL-4 on the

21st day were observed in the vaccinated group, what characterizes anti-rabies vaccine

induced Th2 response and antibody production [26,27]. The production of hydrogen

peroxide associated with higher levels of TNFa serum concentration on the 14th and 21st

days, respectively, suggest that macrophage activity stimulation induced by anti-rabies

vaccine occurs, what is in agreement with the report by Blancou et al. [28] who described

Fig. 7. TNF a serum concentration of different groups and at different moments. Values expressed in Mean of

Ranks.


Table 7

Results of IL-1a, IL-3 and IL-12 in the different groups and moments. Values expressed as median (mean and standard deviation) pg/ml, Botucatu-SP, 2000

Test Day Control control P. acnes Infected Infected P. acnes Infected vaccinated P. acnes

IL 1 a 24 h 29.09 (30.658 þ 26.713) 2406 (47.640 þ 57.315) 50.06 (69.797 þ 20.870) 73.323 (63.890 þ 38.274) 58.63 (68.192 þ 54.853)

7 d 13.92 (33.354 þ 41.940) 34.67 (31.938 þ 22.92) 14.68 (45.802 þ 29.688) 46.99 (32.242 þ 31.093) 33.15 (62.193 þ 44.163)

14 d 22.67 (25.866 þ 26.058) 45.72 (45.725 þ 54.387) 31.54 (38.378 þ 16.577) 40.28 (28.809 þ 15.406) 65.63 (74.528 þ 29.937)

21 d 25.84 (65.526 þ 95.927) 8.58 (15.906 þ 18.713) 38.89 (43.660 þ 17.375) 43.74 (44.687 þ 18.349) 54.93 (58.735 þ 28.821)

IL 3 24 h 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

7 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (10,496 þ 37,847) 0 (0 þ 0)

14 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (39,108 þ 99,688) 0 (0 þ 0)

21 d 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0) 0 (0 þ 0)

IL 12 24 h 9.11 (12.946 þ 11.544) 0 (18.078 þ 40.424) 16.82 (8.601 þ 13.389) 0 (28.334 þ 32.373) 0 (22.472 þ 32.911)

7 d 0 (0.381 þ 0.852) 0 (34.099 þ 49.242) 12.01 (2.073 þ 2.931) 2.07 (59.558 þ 93.984) 3.66 (32.858 þ 51.173)

14 d 36.89 (55.441 þ 70.114) 0 (0 þ 0) 0 (20.447 þ 41.867) 0 (28.772 þ 57.545) 59.68 (77.900 þ 78.157)

21 d 0 (41.64 þ 93.109) 0 (21.229 þ 32.486) 50.78 (8.355 þ 13.733) 0 (52.490 þ 48.846) 0 (13.487 þ 33.0374)

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delayed hypersensitivity in anti-rabies vaccinated mice, and Megid et al. [29] who

observed macrophage activity induced by anti-rabies vaccine in mice on the 6th and 14th

days post vaccination.

Peritonial and splenic macrophage activity induced by P. acnes was verified in P. acnes

control group on the 7th and at 24 h, respectively, associated to higher IL-6 levels

compared to control group what suggests macrophage activity stimulation induced by the

immunomodulator. These results are corroborated by Tizard [30] whot have described

P. acnes activity on macrophages, with IL-1, TNF-a and IL-6 liberation few hours after

administration.

Macrophage activation is response to virus infection observed in the infected group

submitted or not to vaccination and immunomodulation as evidenced by the serum levels

of IL-1a, are in agreement with Marquette et al. [6] who described IL-1a concentration in

central nervous system, in correlation with rabies virus replication.

The greatest spleen macrophage activity correlated with clinical symptoms in the

animals was observed in all infected groups on the 14th day, what suggest a correlation

between macrophagic activity and rabies symptoms. Although the infected vaccinated

P. acnes group was treated with anti-rabies vaccine and P. acnes, which are both

responsible for macrophage stimulation, this group presented lower macrophage activity

associated with lower IL-6 and higher IL-10 levels, compared to the others infected groups

on the 14th day. This group presented the greatest survival rate. As these cytokines can

inhibit macrophage function [27], it may be considered that protection against rabies may

be connected to the suppression of macrophage activity.

The role of macrophages on rabies pathogenesis has been discussed by several

researchers and associated with mononuclear cell infiltration, production of nitric

Fig. 8. IL-1a concentration of different groups and at different moments. Values expressed in Mean of Ranks.


oxide [31] and toxicity in central nervous system [32]. Higher levels of brain IL-1a

associated with clinical symptoms, and correlated with the induction of ACTH release and

glucocorticoid production with consequent immunossupression observed in rabies was

described by Marquette et al. [6]. Additionally, higher concentration of brain TNF-a was

described by Marquette et al. [7], and correlated with severe brain inflamatory response

and rabies encephalopathy, with consequent neuronal dysfunction.

Higher levels of IL-2 at 24 h associated to higher IL-10 and lower IL-6 levels at all

times, together with the greater survival rate, were observed in the infected, vaccinated

P. acnes treated group, compared to the other infected groups. The levels of IL-2 and IL-

10 may have induced by the anti-rabies vaccine, once their levels were greater than in the

not vaccinated group. Although the vaccinated group presented higher IL-6 leves

indicating cytokine induction by anti-rabies vaccine, the infected vaccinated P. acnes

group presented the lowest concentration of this cytokine, only similar to control groups,

suggesting IL-6 inhibition induced by anti-rabies vaccine, rabies infection and P. acnes

association.

These observations suggested that the association of P. acnes and anti-rabies vaccine

induced a stronger antigenic stimulus with consequent IL-2 production, what may lead to

Natural Killer and Lymphokine-Activated Killer (LAK) cells stimulation, CTL activation

and T cells proliferation [26]. In previous trials it was observed that Natural Killer cells

(NK) stimulation induced by P. acnes in mice infected by rabies virus was correlated with

higher TNF-a serum concentration [18]. Inversely, when anti-rabies vaccine was

associated with P. acnes as post-exposure treatment, the activity of NK cells was lower

than in the group treated only with P. acnes, but it was higher than in the infected group,

suggesting Natural Killer activity stimulation induced by P. acnes and probably CTL cells

activation, induced by anti-rabies vaccine [33].

IL-10 can be induced by bacterial lipopolysaccharide [34], but in the present trial,

similar levels of this cytokine in the groups submitted or not to P. acnes were observed.

IL-10 concentration was directly related to anti-rabies vaccination, once it was greater in

vaccinated groups. This suggests a Th2 response.

Although similar leves of anti-rabies serum neutralizing antibodies were observed in

the infected groups, the higher IL-10 levels observed in the infected vaccinated P. acnes

group could be responsible for most immunoglobulins isotypes produced, which could, in

their twin, be responsible for better serum neutralizing activities, as report by Bancherau

et al. [35].

Production of IL-10 by macrophages infected with virulent strains of Mycobacterium

tuberculosis, with subsequent switch from Th1 cell mediated to Th2 immune response was

described and correlated with the persistence of the intracellular bacteria and formation of

granulomatous lesion [36]. The action of IL-10 on Th1 response may explain the low IL-6

levels observed in the infected, vaccinated P. acnes group suggesting suppression of this

cytokine when anti-rabies vaccine and P. acnes were associated as post-exposure anti-

rabies treatment.

Several trials with HIV (Human Immunodeficiency Virus) have demonstrated that

IL-10 inhibits TNF-a and IL-6 production by infected monocytes. These cytokines are

responsible for facilitating HIV virus replication [37,38]. It may be hypothesize that the

same mechanisms involved in HIV replication may be occur in rabies virus, once virus


infection of macrophage, replication and persistence was reported by Ray et al. [4] and

King et al. [2], and correlated with a longer incubation period with possibility of

subsequent virus replication and productive infection induced by cytokine. This possibility

may be reinforced by the observation of higher percentile of positivity in FAT in several

lymphoid tissues, together with low virus titer in brain of mice infected by rabies,

vaccinated and treated using P. acnes described in a previous trial [33].

The results demonstrated that the association between P. acnes and Fuenzalida-

Palacios anti-rabies vaccine in mice infected by rabies virus induce a higher levels of

IL-10 and low levels of IL-6 aspects correlated to higher percentage of survival in rabies.

Acknowledgements

Financial support by FAPESP.

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