Cytokines in sepsis due to Candida albicans and in bacterial sepsis.

ARTICLE

INTRODUCTION

Sepsis is a major clinical problem, with a mortality of up to 70% among patients with septic shock [1]. Besides bacterial pathogens, Candida albicans is increasingly responsible for nosocomial sepsis, septic shock, and lethal disseminated infection in the critically ill [2, 3]. This increasing frequency of serious Candida infections has multifactorial causes, including more intense therapeutic efforts involving intravascular catheters, broad-spectrum antibiotics, extensive surgery and immunosuppression for neoplastic disease or allograft preservation [2, 4].

During sepsis, proinflammatory cytokines produced by the host initiate a cascade of events resulting in lethal hypotension and irreversible tissue injury [5, 6]. Tumor necrosis factor-alpha (TNF-alpha) is considered as the principal mediator in bacterial sepsis and in septic shock. Induced by TNF-alpha, soluble tumor necrosis factor alpha-receptors (TNF-sR) and interleukin-6 (IL-6) modify the response to TNF-alpha during sepsis [7, 8]. Stimulated by these cytokines, endothelial cells express adhesion molecules, e.g. the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, which enhance the extravasation of neutrophils and mononuclear cells into the site of the microbial invasion, and destruction [9]. In contrast to sepsis and septic shock of bacterial origin, the dynamics of cytokines and adhesion molecules during clinical Candida sepsis have not yet been described. Experimentally, C. albicans stimulates the production of cytokines and adhesion molecules [10, 11]. Moreover, dynamic phenotypic switching [12, 13], mimicry of host ligands [14] and receptors, and other virulence factors, including extracellular proteinases, phospholipase, acidic metabolites, and other exotoxins have been recognized as having shock-inducing potential [13]. In most cases, secretion of these substances coincides with the production of specific mannoproteins and other antigens by elongating germ tubes during the yeast-to-hyphal transition [15]. Although these hyphal, phase-specific substances, in turn, stimulate macrophages to produce the pleiotropic cytokine TNF-alpha in response to extracellular hyphae [16], there is evidence that circulating virulence factors, rather than TNF-alpha, may be responsible for the cardiovascular derangement during candidemic shock [17]. To investigate the role of cytokines and adhesion molecules in non-neutropenic patients with sepsis due to C. albicans, we determined the plasma levels of TNF-alpha, IL-6, TNF-sR, VCAM-1 and E-selectin in 20 patients with Candida sepsis, in 20 patients with bacterial sepsis and in 20 non-infectious controls on days 1, 7 and 14.

PATIENTS AND METHODS

The study was performed over the period 1996-1998 at 6 intensive care units of the University Hospital of Vienna, a 2,000 bed referral hospital, and had been approved by the institutional review board. Forty patients and 20 controls were included after giving their informed consent. Patients were prospectively included if [1] either bacteria or C. albicans were isolated from at least two blood cultures, and [2] at least two of the following criteria of sepsis, as defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee [18], were met: a) temperature of > 38° C or < 36° C; b) an increased heart rate of > 90 beats/min; c) respiratory rate of > 20 breaths/min or hyperventilation, as indicated by a PaCO₂ of < 32 torr (< 4.3 kPa); and d) an altered white blood cell count of > 12 G/l, < 4G/l, or the presence of > 10% immature neutrophils ("bands"). Severe sepsis was defined as sepsis associated with organ dysfunction, hypoperfusion abnormality and sepsis-induced hypotension. Hypoperfusion abnormalities included lactic acidosis, oliguria, or an acute alteration of the mental status. Septic shock, as a subset of severe sepsis, was defined as sepsis-induced hypotension (systolic blood pressure < 90 mmHg or its reduction by >= 40 mmHg from the baseline, in the absence of other causes for hypotension), persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction. Parameters for the calculation of the Acute Physiology And Chronic Health Evaluation II (APACHE II) were assessed on inclusion into the study [4].

Patients with neutropenia (< 1,000 G/l), severe hepatopathy, surgery less than 7 days earlier, and those receiving therapy interfering with the production of cytokines (e.g. corticosteroids, pentoxifyllin, anti-cytokine-antibodies etc.) were excluded from the study because of the possible interference and alteration of cytokine levels. Further exclusion criteria were age < 18 years, pregnancy, lactation, advanced malignant disease, or simple catheter-associated infection. Additionally, patients were not included into the study group with Candida sepsis, if they had any clinical or microbiological evidence of a recent or concurrent bacterial infection.

Patients with candidiasis

Twenty patients (15 males, 5 females) with sepsis due to C. albicans were included into the study. The median age was 49 (range 18-81) years. All patients had been admitted to intensive care units on average 11 days earlier (range 6-65 days). On admission, the median APACHE II score was 17.4 (range 14.2-23.3), and 19 patients fulfilled the criteria for sepsis, one of them for severe sepsis. Septic shock was not observed. Underlying diseases included trauma (n = 5), peptic ulcer perforation (n = 3), coronary heart disease (n = 2), transplantation (n = 2), and non-metastatic carcinoma of the lung, rectal carcinoma, arterial occlusive disease, cerebral thrombosis, cholelithiasis, pulmonary fibrosis, aneurysm of the aorta and intravenous drug abuse (one each). Fluconazole 5-10 mg/kg/day was administered to 13 patients, and amphotericin B, 0,7-1 mg/kg/day to 7 patients. Seven patients, 2 receiving amphotericin B and 5 receiving fluconazole (p > 0.05), died within a median of 19 days after inclusion into the study (Table 1).

Patients with bacterial sepsis

Twenty patients (16 males, 4 females) with bacterial sepsis, matched to the patients with Candida sepsis in age and the duration of the ICU admission, were then prospectively included. The causative pathogens isolated from blood were Staphylococcus aureus (n = 9), group A streptococci (n = 4), Escherichia coli (n = 2), Enterobacter cloacae, Salmonella typhi, Salmonella paratyphi, Morganella morgagni and Klebsiella pneumoniae (n = 1, each). The underlying diseases were coronary heart disease (n = 6), peptic ulcer perforation (n = 2), transplantation (n = 2), arterial occlusive disease (n = 2), and chronic pulmonary obstructive disease, chronic renal failure, trauma, non-metastatic breast cancer and intravenous drug abuse (one, each). Three patients did not have any underlying chronic disease. As there was no difference in plasma cytokine and adhesion molecule levels between patients with bacterial Gram-negative sepsis and with Gram-positive sepsis at any time (p > 0.05), they were considered as one group. The median age was 52 years (range 19-79). The median admittance to the ICU was 10 days (range 9-61 days). On admission the median APACHE II score was 18.2 (range 13.4-23.4). There was no significant difference in the APACHE II scores between the patients with bacterial sepsis and the patients with Candida sepsis. On admission, 15 patients fulfilled the criteria for sepsis, five of them those for severe sepsis. Septic shock was not observed. All patients received adequate antibiotic treatment according to the susceptibility pattern of the causative pathogen. Six patients died within a median of 9 days after onset of the infection (Table 1).

Controls

Twenty hospitalized patients with stable coronary heart disease and without infection awaiting coronary angiography (13 males, 7 females; mean age 51 years [range 30-67]) served as controls.

Cytokine and adhesion molecule measurements

On day 1, blood for blood cultures (VITAL automated blood culture system, bioMerieux, Paris, France) was concurrently collected with blood for cytokine and adhesion molecule measurement. Only in patients with positive blood cultures who were eligible for the study, was additional blood drawn for cytokine and adhesion molecule measurements on days 7 and 14, in the morning. During the later samplings, blood was drawn 24 hours after the prior infusion of amphotericin B to minimize the influence of any immunomodulating properties of the drug. All blood samples were collected into sterile, endotoxin-free, EDTA-coated vacutainer tubes (Becton Dickinson, Rutherford, New Jersey), immediately centrifuged at 2,000 x g at 4° for 10 min and stored at ­ 70° C until concurrent measurement of all samples. Plasma concentrations of cytokines were determined by commercially available enzyme-linked-immuno-sorbent-assays (ELISA) using monoclonal antibodies: TNF-alpha (Quantikine, R&D Systems, Minneapolis, sensitivity of 5 pg/ml), IL-6 (Quantikine, R&D Systems, Minneapolis, sensitivity of 0.7 pg/ml) and the 55 kDa protein of the soluble TNF-receptor (Bender MedSystems, Boehringer, Ingelheim, sensitivity of 80 ng/ml). Plasma concentrations of soluble VCAM-1 and the E-selectin were determined by solid phase ELISA (Quantikine, R&D, Minneapolis) with murine monoclonal antibodies against human VCAM-1 and E-selectin. The sensitivity was 100 ng/ml for soluble VCAM-1 and < 0.1 ng/ml for E-selectin. To avoid attenuation of cytokine activity by multiple freeze-thaw cycles, all ELISA tests were run concurrently and in duplicate.

Statistical analysis

All data are presented as the median and the range. All statistical analysis were done with the SAS Statistical Software (Cary, NC). The Kruskal-Wallis, Anova and the Wilcoxon rank-sum test were used for comparing the groups. The Spearman's rank correlation was used to define a correlation between the cytokines and adhesion molecules. The changes of plasma concentrations within groups were calculated with the Friedman-Anova and Wilcoxon signed-ranks test. The level of significance was set at p < 0.05 for the comparisons between two groups, and at p < 0.01 for the correlation coefficient.

RESULTS

Plasma concentrations of TNF-alpha, IL-6 and TNF-sR (55kD protein) were increased in patients with Candida sepsis, and in patients with bacterial sepsis, compared to those of the non-infectious controls (TNF-alpha 2.9 [0-4.9] pg/ml; IL-6 3.4 [0-17.3] pg/ml; TNF-sR 2.0 [0-6.2] ng/ml). TNF-alpha was increased in both sepsis groups on days 1 and 7 (Figure 1). On day 14, TNF-alpha was still increased in patients with Candida sepsis while it was normal in patients with bacterial sepsis (8.0 pg/ml versus 0.8 pg/ml; p < 0.05). IL-6 was increased in both patient groups on day 1 (Figure 1). On days 7 and 14, blood levels of IL-6 were higher in patients with Candida sepsis than in patients with bacterial sepsis (Day 7: 104 pg/ml versus 75.4 pg/ml; day 14: 62.7 pg/ml versus 3.9 pg/ml). TNF-sR was similarly increased in both patient groups and remained elevated throughout the study (Figure 1). IL-6 correlated with TNF-sR on days 7 and 14 (p < 0.005).

The blood concentrations of soluble adhesion molecules were increased in both patients with Candida sepsis and with bacterial sepsis compared to those of the non-infectious controls (E-selectin 48,9 [14,3-89,9] ng/ml; VCAM-1 545 [374-829] ng/ml). Blood concentrations of VCAM-1 were higher in patients with Candida sepsis as compared to patients with bacterial sepsis at any measurement interval (Figure 1; Day 1: 2,633 ng/ml versus 1,548 ng/ml; day 7: 2,225 ng/ml versus 1,235 ng/ml); day 14: 1,832 ng/ml versus 818 ng/ml; p < 0,05). Blood levels of E-selectin were significantly lower in patients with Candida sepsis than the patients with bacterial sepsis (77.5 ng/ml versus 130; p < 0.05) on day 1, only. They decreased significantly over the study period in both patient groups (p < 0.05). VCAM-1 levels correlated with those of TNF-alpha, TNF-sR and IL-6 on days 7 and 14 (p < 0.005, r = 0.485-0.707).

Among the patients with Candida sepsis seven patients died after a median interval of 19 days (range 6-32 days) and six died among the patients with bacterial sepsis after a median interval of 9 days (range 6-29 days) (p not significant). Overall, non-survivors had significantly greater concentrations of E-selectin on days 1, 7 and 14, of TNF-sR and IL-6 on days 1 and 7, than survivors (Table 2). There was no significant difference in TNF-alpha and VCAM-1 concentrations between survivors and non-survivors at any time.

Thirteen patients received fluconazole, and 7 received amphotericin B for the treatment of candidiasis. There was no difference in cytokine and adhesion molecule levels between these two groups at any time.

DISCUSSION

The release of cytokines, particularly of TNF-alpha, was considered to be linked to the release of endotoxin by Gram-negative bacteria [19] and of peptidoglycans released by Gram-positive bacteria [20]. Elevated cytokine levels have also been described in sepsis and malaria (21-23). TNF-alpha and gamma-interferon have been shown to potentiate the growth-inhibiting activity of human neutrophils against C. albicans in vitro [24], and TNF-alpha increased neutrophil fungicidal activity, the production of oxygen radicals, and the release of lysosomal enzymes [25]. In mice, the production of TNF-alpha, as well as IL-6, was induced by C. albicans in normal and neutropenic mice [26, 27]. TNF-alpha production was inoculum dose-dependent [27] and the amount of circulating TNF-alpha was directly related to the inoculum size in both neutropenic and non-neutropenic mice. Further, TNF-alpha played a potentially beneficial role during C. albicans infection [28]. In contrast, TNF-alpha was not detected in septic shock due to C. albicans at an inoculum dose of 10⁹ viable blastoconidia/1 ml in normal or neutropenic rats [17, 29]. Lethal candidemic shock coincided with the yeast-to-hyphal transition in vivo, and in both, lethal shock and hyphal transition preceded increases in circulating blood levels of TNF-alpha. These blood levels, at their peak, were less than 1% of TNF-alpha concentrations during lethal Escherichia coli sepsis. Hence, circulating fungal virulence factors or host mediators other than TNF-alpha were considered to mediate candidemic septic shock [17]. In our study, TNF-alpha, IL-6 and TNF-sR were increased in both, patients with Candida and with bacterial sepsis on day 1. Differences were observed later in the course of sepsis when TNF-alpha and IL-6 decreased to nearly normal in the patients with bacterial sepsis, but remained elevated in the patients with Candida sepsis. In our study, most patients had sepsis, but only one fulfilled the criteria for severe sepsis on admission. In this regard, doubtless, the present study is different to the experimental setting where septic shock was induced by a high inoculum of viable blastoconidia. Thus, as the initial inoculum of the causative pathogen in sepsis cannot be quantified, a comparably low inoculum of C. albicans at the initial phase may stimulate the production of TNF-alpha and the consequent activation of the cytokine cascade. Finally, our observation may be of clinical relevance: the persistant elevation of proinflammatory cytokines in patients with Candida sepsis seems to justify an antifungal therapy for a longer period, although the optimal treatment duration of systemic Candida infections is unknown [30].

Increased concentrations of the adhesion molecules, E-selectin and soluble VCAM-1 were detected in patients with systemic inflammatory response syndrome [31, 32]. The prognosis of septic patients with organ dysfunction was shown to be poor when E-selectin and soluble VCAM-1 remained elevated [33-35]. Transcription and expression of the adhesion molecules, E-selectin and VCAM-1, on endothelial cells were induced by germinating C. albicans, although to a lesser extent than by TNF-alpha [11]. In the present study, the soluble adhesion molecules were increased in patients with Candida sepsis and with bacterial sepsis compared to the control group. On day 1, blood levels of E-selectin were significantly elevated in patients with Candida sepsis compared to controls, but lower than in patients with bacterial sepsis. Later on, there was no difference between the two sepsis groups. Blood levels of VCAM-1 were significantly greater in patients with Candida sepsis than in patients with bacterial sepsis throughout the whole observation period. The production of VCAM-1 and E-selectin by endothelial cells has been described as being induced by adherent C. albicans strains, possibly due to the secretion of aspartyl proteinase or phospholipases [36]. VCAM-1 contributes to the accumulation of lymphocytes and other mononuclear cells at the sites of candidal infection [37]. Although neutrophils are important in the host defense against hematogenously disseminated candidiasis [38], mononuclear cells, particularly T-helper lymphocytes seem to contribute to the defense against this type of infection, and partly compensate neutrophil function in neutropenia [39]. The significant increase of VCAM-1 in patients with Candida sepsis might be due to direct interaction of C. albicans with endothelial cells and may be partly responsible for an enhanced recruitment of mononuclear cells to the site of the fungal infection. Still, it cannot be excluded that the response of endothelial cells to blood-borne microbial pathogens may be additionally influenced by cytokines. Moreover, a variety of host cells, including monocytes/macrophages and bone marrow fibroblasts are able to synthetize VCAM-1, and may be involved in the systemic response during C. albicans sepsis.

Increased blood levels of the cytokines and soluble adhesion molecules were detected in patients with Candida sepsis and in patients with bacterial sepsis. The protracted clinical course of Candida infection and sepsis may explain the still increased TNF-alpha levels on day 14 reflected by the stable increase of TNF-sR. Nevertheless, the possibility of occult bacterial infection or transmigration of bacteria from the gastrointestinal tract into the bloodstream, activating the cytokine cascade can never be excluded during this severe illness. However, bacterial cultures from blood and other sites were negative in the patients with Candida sepsis included in this study. On the other hand, amphotericin B has certain immunomodulating properties resulting in cytokine increase in in vitro and in vivo models [41, 42]. However, in a clinical study in patients with acute myeloic leukemia TNF-alpha, IL-6 and IL-1Ra levels returned to baseline 24 hours after the infusion. The increase of these cytokines in response to amphotericin B was highly variable, depending on the preparation of amphotericin B [43]. In our study, cytokine and adhesion molecule levels were not different between patients receiving amphotericin B and patients receiving fluconazole.

Plasma levels of cytokines and adhesion molecules were prognostic factors in patients with sepsis and systemic inflammatory response syndrome [32, 33, 44]. In agreement with these previous findings, non-survivors in our study had elevated concentrations of E-selectin throughout the study, and of TNF-sR and IL-6 on days 1 and 7. Differences between survivors and non-survivors regarding these cytokines may not be detectable at day 14, probably because the results from patients who had died were missing.

CONCLUSION

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