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CORRELATION BETWEEN SERUM LEVELS OF

INTERLEUKIN-1β, INTERLEUKIN- 1RA,

INTERLEUKIN-6, INTERLEUKIN-10, INTERLEUKIN 12,

TUMOR NECROSIS FACTOR-α AND INTERFERON-γ

WITH SOME CLINICAL AND LABORATORY

PARAMETERS IN PATIENTS WITH SALMONELLOSIS M.S. Stoycheva1, M.A. Murdjeva2 University Hospital “St. George”, Department of Infectious Diseases, Plovdiv, Bulgaria1 University Hospital “St. George”, Department of Microbiology and Immunology, Plovdiv, Bulgaria 2

ABSTRACT Salmonella bacteria are the major cause of food-borne infectious diarrhea. There is great interest in understanding the pathogenesis and immunogenesis of Salmonella infection. Cytokines are key communicator molecules between host cells and the enteric pathogen Salmonella. Serum levels of IL-1, IL-1ra, IL-6, IL-10, IL-12, TNF-α and IFN-γ were studied in a prospective controlled study including 36 patients at the acute stage of gastro-intestinal salmonellosis. Correlation between them was investigated, as well as between cytokines, body temperature and serum levels of Na+, K+ and Cl-. The lowest correlation was found between TNF-α and IL-1ra. A significant and directly proportional correlation was established between IL-12 with IFN-γ and TNF-α, as well as in TNF-α/IL-1β and IL-1β/IL-1ra. The increased body temperature correlated significantly with IL-1β, TNF-α and IL-6. Significant but converse was the correlation between Na+ with IL-1β and IL-6, as well as between body temperature with Na+, K+ and Cl-. Our data imply evidence for the complicated network in which cytokines act with multiple potential synergistic and antagonistic effects between them participating in the main pathogenetic processes – in-toxication and loss of fluids and electrolytes. Introduction Cytokines are involved in initiation, ampli-fying, perpetuating and resolving the in-flammatory and immune responses. They are key mediators for tissue damage and play an important role in many systemic disease manifestations (25).

Abbreviation: Interleukin-1β (IL-1β), Interleukin-1ra (IL-1ra), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin-12 (IL-12), Tumor Necrosis Factor – α (TNF-α), Interferon-γ (IFN-γ)

Cytokines are classified as pro-inflam-matory (TNF-α, IL-1, IL-2, IL-6, IL-8, IFN-γ) and anti-inflammatory (IL-4, IL-10, IL-13). The pro-inflammatory ones are “alarm” cytokines, inducing vascular dila-tation with increased permeability and en-hanced adherent ability of endothelial cells. These cytokines enable proliferation and hemotaxis of polymorphonuclear leuko-cytes to the place of injury and lead to fur-ther tissue damage (10,21,28,30). They also stimulate the pituitary gland for releasing

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stress hormones and the hepatocytes for acute-phase protein synthesis (28). The acute-phase response includes as well mechanisms for control upon the inflam-matory reaction – secretion of anti-inflam-matory cytokines, receptor antagonists and cortisol.

The insufficient cytokine production or the inability for adequate response to them may lead to immune suppression, but their excessive increase – to shock and autoim-mune disorders (12,16,25,28). Recently the pathological events, mediated by cytokines, have been connected mainly with imba-lance in production of pro-and anti-in-flammatory ones. The ration between them is proportional to the influence for their production (15,31,38). When the cytokines act mainly locally in the inflammation site (in auto- or paracrine manner), the favor-able effects for the body dominate. They are directed to stimulation of defense mechanisms. On the contrary, extremely elevated serum concentrations result in prevalence of their destructive effects (4,21). According to others, neither peak, nor mean serum levels are decisive for the degree of damage but the time duration for cytokine increase (24).

In Salmonella infection the main activa-tor of cytokine induction is the lipopoly-saccharide (endotoxin) with marked mul-tipotent ability for noncytotoxic interaction with host cells and tissues and their activation (2, 20, 25, 35). Salmonella porines and flaggelins are other activators too (5,13,37).

Some experimental works report that Salmonella spp. as intracellular pathogens induce Th1 related inflammatory response (14, 23, 31) and the tissue alteration is pre-ceded by release of pro-inflammatory cy-tokines (22). In patients with salmonellosis, the type of cytokine response and the im-portance of cytokine for the course and outcome of the disease, are still unclear. Our recent investigations showed that IL-1β and IL-ra serum levels were increased in these patients during acute and conva-

lescent stage of salmonellosis (29). The aim of this study was to investigate

the correlation between serum levels of IL-1β, IL-1ra, IL-6, IL-10, IL-12, TNF-α and IFN-γ; the febrile reaction and serum levels of Na+, K+ and Cl- in patients during acute stage of salmonellosis.

Materials and Methods The study included 36 patients (aged 19-56 years) with gastrointestinal salmonellosis. They were treated at the Clinics of Infec-tious Diseases, University Hospital – Plovdiv. All patients had culture confirmed diagnosis. S. enteritidis was the etiological agent in 81% of the cases, S. typhimurium – in 13,5% and other Salmonella serotypes - in 5.5%.

Investigations were performed during acute disease stage. Blood samples for electrolytes and cytokines were collected upon admission and before treatment ini-tiation. Serum levels of Na+, K+ and Cl- were detected in arterialized capillary blood with an automated analyzer (288 Blood-Gas System, Ciba Corning). The analysis of electrolytes was performed im-mediately after the blood collection.

Cytokine levels were measured from sera using ELISA kits (BioSource, Medgenix Diagnostics, Fleurus, Belgium) according to manufacturer’s instructions and as des-cribed briefly before (29). Serum concen-trations were determined for all cytokines in one experiment. The sera were frozen at –70 ºC for no longer than 6 months and then thawed at the day of testing.

The body temperature of all patients was appreciated as the main component of toxic infectious syndrome, and the electrolyte disorders as the most important markers for gastro-enteric dysfunction in salmonellosis.

Descriptive statistics and correlation analysis using Microsoft Excel 2000 were applied. The degree of correlation was as-sumed on the basis of Pearson coefficient (rxy).The correlation was significant at 0.01 level (2-tailed).

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Results and Discussion Correlation between serum levels of tested cytokines. A high and directly pro-portional correlation was observed between TNF-α and IL-ra. This confirms the data by others (1,32), who suggested that TNF-α together with IL-1 and the Salmonella en-dotoxin are inducers for IL-ra production. Probably this is a manner to control the inflammatory reaction by blocking the in-flammatory effect of IL-1, which physio-logical inhibitor is IL-1ra.

Our findings showed that a significant correlation existed between serum levels of IL-12 with TNF-α and IFN-γ. This is in concordance with the data of Mizuno J et al (21) in patients with salmonellosis as well as with the results from experimental stu-dies (9,28,34). According to these authors Salmonella spp. induce IL-12 production from macrophages and dendritic cells after internalization into them. IL-12, on its turn, stimulates IFN-γ production from Th1 and NK cells, and TNF-α synthesis from monocytes, macrophages, NK and B cells (8,22,28). IFN-γ with the endotoxin, even in minimal concentrations, induces synthe-sis of IL-12 and regulates its effect upon NK and T cells (6,9,28). IL-12 and IFN-γ mutually amplify their effect which is in-hibited by IL-4 and IL-10 (28,38). IL-12 enables proliferation and functional activity of NK cells and stimulates synthesis of pro-inflammatory cytokines (27). IFN-γ acti-vates macrophages which undergo a num-ber of biochemical and functional changes leading to their ability for cytokine produc-tion (IL-1, IL-6, IL-8, TNF-α) and in-creased cytotoxicity (28).

According to our data, the correlation between TNF-α and IL-1β serum levels, and between TNF-α and IL-6 is also sig-nificant. This finding correlates with the results of other authors, who have estab-lished that IL-1 and IL-6 mediate the pathological effects of TNF-α (14). In pa-tients with typhoid fever, a significant in-crease of TNF-α and IL-6 has been found

and these concentrations correlated with the severity and fatal outcome of the di-sease (3).

A significant directly proportional corre-lation was established in our patients in terms of IL-1β and IL-ra serum concentra-tions. This might be explained with the fact that IL-1 induces the synthesis of its natural inhibitor (2,7,32). The attachment of IL-ra to their common receptor on the target cells switches off the action of IL-1 and protects the body from its harmful effects (10). It is accepted that receptor saturation with IL-ra is possible at ratio 10:1 between IL-ra and IL-1 (7).

The data concerning the significant and directly proportional correlation between IL-1β and IL-10 are intriguing. It is well known that IL-10 is the most potent anti-inflammatory cytokine inhibiting IL-12, IFN-γ, TNF-α and IL-1 synthesis, and re-ducing the capability of target cells for res-ponse to these cytokines (18,19,33). In terms of this fact, the converse correlation between IL-1β and IL-10 would have been more logical. Experimental Salmonella infection in mice has shown dynamics in IL-10 levels, similar with that of pro-in-flammatory cytokines, and providing regu-latory mechanism in vivo on their effect (1,17,18,19,33). Correlation between serum IL-1β, IL-6 levels and electrolytes. The moderate and conversely proportional correlation bet-ween IL-1β and Na+, as well as between IL-6 and Na+ and Cl- in sera of our pa-tients, can be explained with the inflam-matory effect of these cytokines and the great loss of Na+ and Cl- in acute and pro-nounced inflammatory response. Some in-vestigators believe that IL-1 has a crucial role in the origin of diarrheal syndrome in salmonellosis and direct secretory effect on intestinal epithelium (11,26). According to others, this cytokine stimulates target cells to produce prostaglandines and other pro- inflammatory mediators which activate the intestinal epithelial cells (30, 32).

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TABLE Significant correlation between tested cytokines and basic clinical and laboratory parameters

Pearson coefficient (rxy) 0,9 – 0,7 0,7 – 0,5 0,5 – 0,3 Type of proportion direct converse direct converse direct converse

parameters

TNF-α/IL-1ra IL-12/IFN-γ IL-12/TNF-α IL-1β/IL-10 IL-1β/IL-1ra TNF-α/IL-1β

K+/Cl-

IL-1β/t0 TNF-α/ t0 IL-6/ t0 TNF-α/IL-6 IL-6/IL-1ra

IL-10/t0 t0/Na+ t0/K+ t0/Cl- IL-1β/Na+ IL-6/Na+ IL-6/Cl-

Correlation of serum cytokines with the body temperature. Our data established significant and direct correlation between serum IL-1β, TNF-α and IL-6 levels with temperature. IL-1 and TNF are the main endogenous pyrogens. They act synergisti-cally and amplify mutually causing fever, febrile reaction, muscle proteolysis, ano-rexia and hypoglycemia (30).

Cytokines are multifunctional molecules and the actions of some of them overlap to a certain extent. The network and the con-ditions in which they play, are characte-rised with many potentially synergistic and antagonistic effects between them as well as with many possibilities for internal control.

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