Skip to content


  • Research
  • Open Access

Inducibility of the endogenous antibiotic peptide β-defensin 2 is impaired in patients with severe sepsis

  • 1Email author,
  • 1, 2,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1 and
  • 1
Critical Care200711:R19

  • Received: 31 July 2006
  • Accepted: 15 February 2007
  • Published:



The potent endogenous antimicrobial peptide human β-defensin 2 (hBD2) is a crucial mediator of innate immunity. In addition to direct antimicrobial properties, different effects on immune cells have been described. In contrast to the well-documented epithelial β-defensin actions in local infections, little is known about the leukocyte-released hBD2 in systemic infectious disorders. This study investigated the basic expression levels and the ex vivo inducibility of hBD2 mRNA in peripheral whole blood cells from patients with severe sepsis in comparison to non-septic critically ill patients and healthy individuals.


This investigation was a prospective case-control study performed at a surgical intensive care unit at a university hospital. A total of 34 individuals were tested: 16 patients with severe sepsis, 9 critically ill but non-septic patients, and 9 healthy individuals. Serial blood samples were drawn from septic patients, and singular samples were obtained from critically ill non-septic patients and healthy controls. hBD2 mRNA levels in peripheral white blood cells were quantified by real-time polymerase chain reaction in native peripheral blood cells and following ex vivo endotoxin stimulation. Defensin plasma levels were quantified by enzyme-linked immunosorbent assay.


Endotoxin-inducible hBD2 mRNA expression was significantly decreased in patients with severe sepsis compared to healthy controls and non-septic critically ill patients (0.02 versus 0.95 versus 0.52, p < 0.05, arbitrary units). hBD2 plasma levels in septic patients were significantly higher compared to healthy controls and critically ill non-septic patients (541 versus 339 versus 295 pg/ml, p < 0.05).


In contrast to healthy individuals and critically ill non-septic patients, ex vivo inducibility of hBD2 in peripheral blood cells from septic patients is reduced. Impaired hBD2 inducibility may contribute to the complex immunological dysfunction in patients with severe sepsis.


  • Severe Sepsis
  • Septic Patient
  • Peripheral Blood Cell
  • Polymerase Chain Reaction Efficiency
  • hBD2 mRNA


Endogenous antimicrobial peptides are widely distributed in various species [1, 2]. They are part of the innate immune system and their genes are highly conserved throughout the animal and plant kingdoms. In humans, antimicrobial defensins are divided into α- and β-defensins according to their molecular structure. They display a broad antimicrobial effect against bacteria, fungi, mycobacteria, and coated viruses [25]. Defensins act by permeabilising microbial membranes. In addition, β-defensins are chemotactic for immature dendritic cells and memory T cells. They regulate cytokine production and adhesion-molecule expression, stimulate epithelial cell and fibroblast proliferation, and promote histamine release from mast cells [6, 7].

To date, six human β-defensin genes have been characterised and located on chromosome 8. The epithelial human β-defensin 1 (hBD1) gene is constitutively expressed at low levels and slightly upregulated following stimulation [8]. In contrast, hBD2, hBD3, and hBD4 gene expression is inducible mainly by various inflammatory stimuli in different cell types [912] The recently described hBD5 and hBD6 represent epididymis-specific human defensins [13].

There is increasing evidence for the clinical relevance of defensins. Alpha- and β-defensins contribute to anti-HIV activity [14, 15]. In newborns, respiratory tract β-defensin mRNA expression is upregulated in response to infection [16]. Moreover, a systemic release of β-defensins in infectious diseases has been reported [17]. Our own previous experiments detected hBD2 mRNA expression in white blood cells following ex vivo stimulation by endotoxin [18]. In particular, systemic infection underlying syndromes such as severe sepsis challenges the immune system by constant activation of its adaptive and innate components. The responsiveness of the innate immune system, including expression of endogenous antibiotic peptides like β-defensins, contributes to the final resolution of the disease.

The present study investigated hBD2 mRNA levels in native peripheral white blood cells as well as the ex vivo hBD2 mRNA inducibility in patients with severe sepsis. Additionally, we determined hBD2 protein plasma levels in patients. The hypothesis that hBD2 expression is disturbed in patients with severe sepsis was tested.

Materials and methods

Patients and controls

This study was performed according to the ethical standards stated in the 1964 Declaration of Helsinki. After approval by the local ethics committee and receipt of the written informed consent of either the patient or a close relative, 16 patients treated on a surgical intensive care unit (ICU) at a university hospital with the diagnosis of severe sepsis were included in this prospective case-control study. The diagnosis of severe sepsis met the criteria of the American College of Chest Physician/Society of Critical Care Medicine Consensus Conference Committee [19]. Exclusion criteria were (a) lack of informed consent, (b) age younger than 18 years, and (c) pre-existing immunological or haematological diseases. Whole blood samples were drawn on the day of diagnosis (day 1) and on the third and fifth days of severe sepsis. A fourth blood sample was drawn after recovery from severe sepsis at ICU discharge in survivors or at imminent death in the case of non-survivors (day X).

In addition, two control groups were included: nine non-septic critically ill ICU patients who were in need of intensive care and who were without any signs of infection (blood samples were drawn once during the ICU treatment) and nine healthy volunteers (blood samples were drawn once). All patients and volunteers were of German Caucasian origin.

Blood culture and RNA isolation

Whole blood was co-cultured for four hours with 500 pg/ml lipopolysaccharide contained in the Milenia® ex vivo stimulation kit (Milenia Biotec, Hohe Str. 4–8, 61231 Bad Nauheim, Germany) at 37°C and 5% CO2. After incubation, the blood was centrifuged at 1,500 g for five minutes. The supernatant was stored at -70°C for further analysis. Total RNA was extracted from whole blood by means of a QIAamp® RNA Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and then dissolved in diethylpyrocarbonate-treated water and stored at -70°C until further analysis.

Basic hBD2 mRNA levels were investigated using Paxgene® Blood RNA System tubes (PreAnalytiX; Qiagen GmbH, Hilden, Germany). For this analysis, 2.5 ml of whole blood was drawn in Paxgene® tubes and treated as indicated in the manufacturer's instructions. By this method, intracellular RNA was stabilised until further analysis. RNA isolation was performed using the Paxgene® kit according to the manufacturer's instructions.

cDNA preparation

cDNA was produced as polymerase chain reaction (PCR) template using 1st Strand cDNA Synthesis Kit for RT-PCR® (avian myeloblastosis virus [AMV]) (Roche Diagnostics, Sandhofer Str. 116, 68305, Mannheim, Germany). The reaction mixture contained 8.2 μl (approximately 500 ng) of total RNA, 5 mM MgCl2, 1 mM dNTP, 3.2 μg of random primer p(dN)6, 50 units of RNase inhibitor, 20 units of AMV reverse transcriptase, and 1× reaction buffer in a total volume of 20 μl. The reaction was incubated at 25°C for 10 minutes, 42°C for 60 minutes, and 99°C for 5 minutes and then cooled to 4°C for 5 minutes.

Real-time PCR

The PCR was performed on a LightCycler® instrument (Roche Diagnostics). For the amplification of hBD2, the reaction mixture included 10 μl of cDNA, 1 μM each primer (forward and reverse), 0.15 μM each hybridisation probe (labelled with fluorescein and LC-Red640; TIB MOLBIOL GmbH, Berlin, Germany), and 1× Lightcycler FastStart MasterPLUS Mix (Roche Diagnostics) in a total volume of 20 μl. For detection of the housekeeping gene hHPRT (human hypoxanthine phosphoribosyl-transferase), the 20 μl of reaction mixture consisted of 2 μl of cDNA, 2 μl of reaction mix for hHPRT (Roche), and 12 μl of ddH2O in 1× Lightcycler FastStart MasterPLUS Mix (Roche Diagonostics). The sequences of primers and hybridisation probes specific for hBD2 measurement were as follows: forward primer: 5'-CTGATGCCTCTTCCAGGTGT-3'; reverse primer: 5'-GGAGCCCTTTCTGAATCCG-3'; probes: 5'-GGTATAAACAAATTGGCACCTGTGGTC-FL and 5'-LC Red640 CCCTGGAACAAAATGCTGCAAAA-PH.

The PCRs for hBD2 and hHPRT were conducted in separate capillaries as duplicates. The reaction was performed as follows: initial denaturation at 95°C for 10 minutes followed by 45 cycles of 95°C for 5 seconds, 55°C for 8 seconds, and 72°C for 10 seconds. The reaction was then cooled at 40°C for 30 seconds. Fluorescence was monitored at the end of each 55°C incubation and detected in channel F2/F1. The crossing point (CP) of each reaction was analysed by the method of second derivative maximum algorithm (CP was defined as cycle number at detection threshold).

Relative quantification analysis

The expression level of hBD2 mRNA in each sample was analysed by LightCycler Relative Quantification Software (Roche Diagnostics). The principles and workflows have been described previously [20]. In summary, the quantity of a target (hBD2) and a reference (hHPRT) gene is a function of the PCR efficiency and the sample CP and does not require a standard curve in each LightCycler analysis run for its determination. CP value is most reliably proportional to the initial template concentration. Differences in PCR efficiency result from different primers as well as hybridisation probes and can be corrected by the software. Results are expressed as the target/reference ratio of each sample normalised by the target/reference ratio of the calibrator. The calibrator is included in every run and its ratio is set to a value of 1. This normalisation provides a constant calibrator point between PCR runs.

Normalised ratio = conc .target  ( sample ) con .reference  ( sample ) : conc .target  ( calibrator ) conc .reference  ( calibrator ) , MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=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@BA81@

Normalised ratio = ETCpT(C) - CpT(S) × ERCpR(S) - CpR(C),

where E = efficiency of PCR amplification, T = target gene, R = reference gene, S = unknown sample, and C = calibrator.

In this experiment, the coefficient file was created by PCR amplification of hBD2 and hHPRT as the housekeeping gene in a series of diluted cDNA (relative standard curve) in triplicates. Data of real-time PCR, including calibrator and samples, were imported into the Relative Quantification Software and analysed with the Fit Coefficients File. Finally, the normalised ratios were calculated. These ratios directly reflect the expression level of hBD2 mRNA.

hBD2 plasma protein quantification

Twenty micrograms of hBD2 polypeptides was diluted in acetic acid to form the 1 μg/μl stock solution and then adjusted to 10 mM Tris/0.5% bovine serum albumin (BSA)/0.05% Tween-20 to obtain serial concentrations of the hBD2 standard: 2,000 pg/ml, 1,000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, and 62.5 pg/ml. Samples were diluted in 1:4 dilution buffer 10 mM Tris/0.5% BSA/0.05% Tween-20. Coating of the standards and samples was performed in a 96-well plate with 100 μl of phosphate-buffered saline coating buffer at 4°C overnight.

Thereafter, the plates were blocked with 300 μl of 5% non-fat bovine milk blocking buffer at 37°C for 2 hours. The goat polyclonal β-defensin 2 antibody (Abcam plc, 332 Cambridge Science Park, Milton Road, Cambridge, UK) was diluted to 0.5 μg/ml with 5% non-fat bovine milk antibody dilution buffer. One hundred microlitres was applied to each well. After additional washing, the peroxidase-conjugated rabbit anti-goat immunoglobulin G antibody (1:1,200) (Sigma-Aldrich Chemie GmbH, Eschenstrasse 5, 82024 Taufkirchen, Germany) was applied to the wells. Plates were covered and incubated at 37°C for two hours. Washing was followed by the addition of 100 μl of ready-to-use tetramethylbenzidine substrate to each well. The plate was then covered and incubated at room temperature for 0.5 hours. One hundred microlitres of stop solution was added to each well. Absorbance was measured at 405 nm using a microtiter plate spectrophotometer followed by an endpoint measurement within one hour.

Human leukocyte antigen-DR quantification on circulating monocytes

Flow cytometric human leukocyte antigen-DR (HLA-DR) quantification was performed according to the method of Docke and colleagues [21]. In brief, this new method quantifies the number of molecules per monocyte and allows direct comparisons between laboratories.

Whole blood cell counts

Leukocyte and monocyte cell counts in whole blood were quantified routinely by standardised clinical biochemical methods.

Statistical analysis

Significance levels between groups were examined using the Kruskal-Wallis test with the Dunn multiple comparison test and Mann-Whitney U test where indicated. A p value of less than 0.05 was regarded as statistically significant. The time course of the Sepsis-related Organ Failure Assessment (SOFA) scores was analysed by two-way analysis of variance (ANOVA) with repeated measures and Bonferroni post hoc analysis. Two-way ANOVA with repeated measures was also used for the time course of hBD2 plasma levels. In contrast, the non-gaussian distribution of ex vivo inducible defensin mRNA expression was analysed by the Kruskal-Wallis test. Correlation of the scores with hBD2 inducibility was tested using the Spearman test. Statistical power calculations were performed using an open-access statistical web page [22].


Sixteen patients with severe sepsis were included in this study. Eight of these patients died from sepsis-induced organ failure. In addition, nine critically ill but non-septic ICU patients and nine healthy volunteers were included. Table 1 shows demographic and clinical data of the patients. Acute Physiology and Chronic Health Evaluation II (APACHE II) and Simplified Acute Physiology Score II scores differed between critically ill non-septic patients and patients with severe sepsis (p < 0.05, Mann-Whitney test), whereas age did not (p > 0.05, Mann-Whitney test). Underlying diseases for severe sepsis were necrotising fasciitis (n = 2; at inclusion, both showed clinical signs of additional pulmonary infection), faecal peritonitis (n = 8), and pneumonia (n = 6). Finally, all patients with severe sepsis suffered from abdominal or pulmonary infection.
Table 1

Demographic and clinical data of critically ill non-septic patients and patients with severe sepsis


Critically ill non-septic (n = 9)

Severe sepsis (n = 16)


Median age (years)



> 0.05

Median APACHE II score at inclusion



< 0.05

Median SAPS II score at inclusion



< 0.05

Mechanically ventilated at inclusion (n)




Vasopressor treatment at inclusion (n)




Median IL-6 plasma levels (ng/l)



< 0.05

Median procalcitonin plasma levels (μg/l)



< 0.05

Antibiotic treatment at inclusion (n)




Statistical differences were calculated by Mann-Whitney test. APACHE II, Acute Physiology and Chronic Health Evaluation II; IL-6, interleukin-6; SAPS II, Simplified Acute Physiology Score II.

Eight of the nine critically ill non-septic patients were in the perioperative period after trauma, abdominal or pharyngeal cancer, or aortic aneurysm rupture with a prolongated postoperative recovery. All of these patients except one were treated with perioperative antibiotic prophylaxis. One patient from this control group suffered from abacterial pancreatitis without antibiotic therapy. None of these patients showed clinically or laboratory signs of infection.

None of the critically ill patients was treated with hydrocortisone. In contrast, 11 patients with severe sepsis were medicated with low-dose hydrocortisone (3 mg/kg body weight per day) at at least one measuring point. All patients with sepsis were treated according to guidelines issued by the Surviving Sepsis Campaign [23].

SOFA score was determined at every time point of blood drawing in the included patients, and APACHE II score was calculated at inclusion. The score differences between the patient groups are illustrated in Table 1. Neither the hBD2 inducibility nor the protein levels showed correlations with APACHE II or SOFA scores (p > 0.05, Spearman test; data not shown). hBD2 plasma levels did not show a correlation with the Horowitz quotient, thrombocyte count, creatinin levels, or the need of use of vasopressors (p > 0.05, Spearman test; data not shown).

SOFA scores in survivors of severe sepsis were decreased at day five and the last sampling day compared to non-survivors (p < 0.05, two-way ANOVA with repeated measures and Bonferroni post hoc analysis; data not shown).

Basic hBD2 mRNA expression was not detectable in peripheral blood cells from healthy controls. The basic hBD2 mRNA expression in survivors and non-survivors of severe sepsis and critically ill patients was normalised to the leukocyte count of every blood sample and showed no differences (p > 0.05, Kruskal-Wallis test with the Dunn multiple comparison test; Figure 1).
Figure 1
Figure 1

Basic human β-defensin 2 (hBD2) mRNA expression normalised to leukocyte count in critically ill non-septic patients and survivors and non-survivors of severe sepsis shows no differences. No basic mRNA expression was detected in healthy controls (p < 0.05, Kruskal-Wallis test with the Dunn multiple comparison test). Data are presented as box-and-whisker plots.

In contrast, hBD2 mRNA was detectable in ex vivo stimulated cultured whole blood. Endotoxin stimulation (4 hours, 0.5 ng/ml) induced hBD2 mRNA expression in all groups and led to low inducibility in patients with severe sepsis. Figure 2 indicates the inducible mRNA expression normalised to leukocyte count at all measured time points. The inducibility in patients with severe sepsis was significantly decreased compared to both other groups (p < 0.05, Kruskal-Wallis test with the Dunn multiple comparison test) without differences between survivors and non-survivors of severe sepsis. Despite the limited number of patients, the statistical power of the comparison of hBD2 mRNA inducibility between patients with severe sepsis and controls was 0.95. Hydrocortisone treatment did not impair the leukocyte count-normalised hBD2 mRNA inducibility in patients with severe sepsis (p > 0.05, Kruskal-Wallis test with the Dunn multiple comparison test; Figure 3).
Figure 2
Figure 2

Ex vivo human β-defensin 2 (hBD2) inducibility in healthy controls, critically ill non-septic patients, and survivors and non-survivors of severe sepsis. Inducible hBD2 mRNA expression normalised to leukocyte count is decreased in survivors and non-survivors of severe sepsis compared to healthy controls and critically ill non-septic patients (*p < 0.05, Kruskal-Wallis test with the Dunn multiple comparison test). Data are presented as box-and-whisker plots.

Figure 3
Figure 3

Ex vivo human β-defensin 2 (hBD2) inducibility in patients with severe sepsis. Inducible hBD2 mRNA expression normalised to leukocyte count shows no differences in cortisone-treated or non-cortisone-treated patients (p > 0.05, Kruskal-Wallis test with the Dunn multiple comparison test). Data are presented as box-and-whisker plots.

In addition, hBD2 protein concentration was quantified in plasma at all included time points. hBD2 plasma concentrations in non-septic critically ill patients and healthy controls were significantly lower compared to patients with severe sepsis (p < 0.05, Kruskal-Wallis test with the Dunn multiple comparison test; Figure 4). The comparison of hBD2 plasma levels reached statistical significance at a power of 0.98. No differences were detected between survivors and non-survivors of severe sepsis.
Figure 4
Figure 4

Human β-defensin 2 (hBD2) plasma protein concentration in healthy controls, critically ill non-septic patients, and patients with severe sepsis. Plasma concentration in healthy controls and critically ill non-septic patients was significant lower compared to patients with severe sepsis (*p < 0.05, Kruskal-Wallis test with the Dunn multiple comparison test). Data are presented as box-and-whisker plots.

hBD2 protein levels showed no correlation with interleukin (IL)-6 plasma levels in septic patients (p > 0.05, correlation coefficient r = -0.041, Spearman test; data not shown). In contrast, procalcitonin (PCT) plasma levels and hBD2 protein plasma levels showed a positive correlation in patients with severe sepsis (p < 0.005, correlation coefficient r = 0.4203, Spearman test; Figure 5).
Figure 5
Figure 5

Human β-defensin 2 (hBD2) plasma protein and procalcitonin (PCT) levels showed a significant correlation in patients with severe sepsis (p < 0.005, Spearman test).

The time course of hBD2 plasma protein concentration in patients with severe sepsis did not differ significantly between survivors and non-survivors, however it showed considerable variation between survivors and non-survivors (p > 0.05, two-way ANOVA with repeated measures; Figure 6).
Figure 6
Figure 6

Human β-defensin 2 (hBD2) plasma protein concentration at different time points in patients with severe sepsis. Time course of hBD2 plasma protein concentration in survivors and non-survivors of severe sepsis showed no statistical differences (p > 0.05, two-way analysis of variance with repeated measures). Data are presented as mean ± standard error of the mean.

HLA-DR quantification was performed in patients with severe sepsis and non-septic critically ill patients. HLA-DR molecules on circulating monocytes per cell in non-septic critically ill patients were significantly higher compared to patients with severe sepsis (p < 0.05, Mann-Whitney U test; data not shown).


The present investigation shows the novel finding of impaired hBD2 gene inducibility in peripheral cells and elevated plasma protein concentration in patients with severe sepsis compared to non-septic critically ill patients and healthy controls. The meaning of β-defensins for the defence of infections is based on well-described antimicrobial activities [24, 25]. In addition, β-defensins induce prostaglandin D2 production, degranulate mast cells, and present chemotactic activities on CCR6-positive dendritic cells [6, 26]. In mice, additional immunomodulatory effects have been reported [6, 27, 28]. These data indicate their involvement in innate immunity. These reported effects suggest regulatory or mediatory defensin functions. The role of antibiotic peptides in the pathogenesis of Crohn's colitis, cystic fibrosis, and panbronchiolitis has been described clearly. An effective defence related to levels and inducibility of defensins has been reported [17, 2933].

The elevated plasma levels of hBD2 in patients with severe sepsis indicate a higher activity of inflammation compared to non-septic individuals. Proinflammatory cytokines such as IL-1 and tumour necrosis factor induce hBD2 gene expression in alveolar macrophages and monocyte-derived epidermis cells (IL-1) [10, 12]. These proinflammatory cytokines, which are frequently elevated in severe sepsis, are potentially involved in the upregulation of systemic hBD2 release in sepsis as well. The decreased hBD2 inducibility in peripheral blood cells was not associated with decreased plasma levels, suggesting that peripheral blood cells do not represent the exclusive source of released hBD2 protein in vivo.

The hBD2 plasma concentration in healthy controls agrees with findings from other investigations [17, 34]. It should be taken into account that circulating endothelial cells or reticuloendothelial cells also represent a possible source of hBD2 [35]. The results for hBD2 mRNA inducibility and the basic protein plasma levels showed no significant differences between healthy controls and critically ill non-septic patients. Median PCT levels were in normal range, indicating a lack of systemic infection, whereas a median IL-6 of 18 ng/l (normal is below 15 ng/l) suggested minor systemic inflammatory activation in the non-septic critically ill patient group. For gene activation of hBD2 and IL-6, the transcription factor nuclear factor-kappa B (NF-κB) is crucial. The low IL-6 levels in the critically ill non-septic group provide a hint for, but are not proof of, low NF-κB activation in this group. This minor activation showed no influence on hBD2 inducibility or protein levels compared to healthy controls. Only the systemic infection in the severe septic patient group led to changes in gene inducibility and plasma levels. These results underline a specific impact of systemic infections on hBD2 gene expression and plasma levels.

The decreased hBD2 mRNA inducibility in peripheral blood cells of patients with severe sepsis could mirror a serious inhibition of innate immune function. But given that the detected plasma concentrations were lower than required for bactericidal/antiviral activity, antimicrobial peptides may not exert their antimicrobial effects via the bloodstream [3638]. However, innate immunity may be impaired not only due to the lack of direct antimicrobial activity but because of limited immunomodulating effects of defensins.

This immunological imbalance occurring in severe sepsis can be monitored, among other ways, by HLA-DR quantification on circulating monocytes. In this manner, the immune competence of monocytes can be assessed. It is well established that monocytes with diminished HLA-DR expression are inhibited in some of their main tasks (for example, antigen presentation and mediator production) [39, 40]. Indeed, the investigated patients with severe sepsis showed signs of immunodepression by decreased HLA-DR expression on circulating monocytes. This finding underlines that sepsis may contribute to the impaired hBD2 inducibility as reported in the present investigation.

In this investigation, hydrocortisone treatment did not impair hBD2 inducibility in patients with severe sepsis. However, at the present time, there are no consistent data on the influence of steroid medication on hBD2 inducibility [4143].

An individual's age can modulate immune function. Activities of cellular components of innate immunity are impaired at different levels [4446]. To date, no data assessing antimicrobial peptide gene expression in the elderly have been collected. However, in insects, antimicrobial peptide gene expression increases with age [47]. The median age of the control group was significantly lower compared to both other groups. There was no significant difference of the median age between the critically ill and the septic patients. Therefore, the differences between the critically ill and the septic patients concerning hBD2 mRNA inducibility and plasma levels cannot be explained by differences in age.


hBD2 inducibility in leukocytes from patients with severe sepsis is decreased. This special part of innate immunity is influenced by severe sepsis. The downregulation of inducibility may contribute to the complex immunological imbalance occurring in patients with severe sepsis.

The importance of plasmatic hBD2 for patients with severe sepsis is unclear. In particular, knowledge of the interaction with mediators and effectors of the immune system is scarce but of prime importance. To date, the antimicrobial and immunomodulatory activities of hBD2 have been tested only in ex vivo settings with limited numbers of additional co-factors. However, in vivo, hBD2 is an integral component of a set of effectors that function together in the innate immune line of defence.

Key messages

  • Ex vivo endotoxin hBD2 inducibility in leukocytes was decreased in patients with severe sepsis compared to healthy controls and critically ill non-septic patients.

  • hBD2 plasma levels were elevated in the severe sepsis group compared to both other groups.

  • hBD2 inducibility and plasma levels showed no differences between survivors and non-survivors of severe sepsis.



avian myeloblastosis virus


analysis of variance


Acute Physiology and Chronic Health Evaluation II


bovine serum albumin


crossing point


human β-defensin 2


human hypoxanthine phosphoribosyl-transferase


human leukocyte antigen-DR

ICU : 

intensive care unit



NF-κB : 

nuclear factor-kappa B


polymerase chain reaction




Sepsis-related Organ Failure Assessment.



The authors thank Angelika Zoons for excellent technical assistance with hBD2 enzyme-linked immunosorbent assay. This study received financial support from the German Research Foundation (BO 1929/1-1).

Authors’ Affiliations

Department of Anaesthesiology and Intensive Care Medicine, Rheinische-Friedrich-Wilhelms University Bonn, Sigmund-Freud-Str. 25, Bonn, 53105, Germany
Department of Anaesthesiology, School of Medicine, Zhejiang University, 388 Yuhang Tang Road, Hangzhou, 310058, People's Republic of China


  1. Lehrer RI, Ganz T: Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 1999, 11: 23-27. 10.1016/S0952-7915(99)80005-3View ArticlePubMedGoogle Scholar
  2. Boman HG: Antibacterial peptides: basic facts and emerging concepts. J Intern Med 2003, 254: 197-215. 10.1046/j.1365-2796.2003.01228.xView ArticlePubMedGoogle Scholar
  3. Miyasaki KT, Bodeau AL, Selsted ME, Ganz T, Lehrer RI: Killing of oral, gram-negative, facultative bacteria by the rabbit defensin, NP-1. Oral Microbiol Immunol 1990, 5: 315-319.View ArticlePubMedGoogle Scholar
  4. Porro GA, Lee JH, de Azavedo J, Crandall I, Whitehead T, Tullis E, Ganz T, Liu M, Slutsky AS, Zhang H: Direct and indirect bacterial killing functions of neutrophil defensins in lung explants. Am J Physiol Lung Cell Mol Physiol 2001, 281: L1240-L1247.PubMedGoogle Scholar
  5. Thevissen K, Francois IE, Takemoto JY, Ferket KK, Meert EM, Cammue BP: DmAMP1, an antifungal plant defensin from dahlia ( Dahlia merckii ), interacts with sphingolipids from Saccharomyces cerevisiae . FEMS Microbiol Lett 2003, 226: 169-173. 10.1016/S0378-1097(03)00590-1View ArticlePubMedGoogle Scholar
  6. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, et al.: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999, 286: 525-528. 10.1126/science.286.5439.525View ArticlePubMedGoogle Scholar
  7. Yang D, Biragyn A, Kwak LW, Oppenheim JJ: Mammalian defensins in immunity: more than just microbicidal. Trends Immunol 2002, 23: 291-296. 10.1016/S1471-4906(02)02246-9View ArticlePubMedGoogle Scholar
  8. Bajaj-Elliott M, Fedeli P, Smith GV, Domizio P, Maher L, Ali RS, Quinn AG, Farthing MJ: Modulation of host antimicrobial peptide (beta-defensins 1 and 2) expression during gastritis. Gut 2002, 51: 356-361. 10.1136/gut.51.3.356PubMed CentralView ArticlePubMedGoogle Scholar
  9. Garcia JR, Krause A, Schulz S, Rodriguez-Jimenez FJ, Kluver E, Adermann K, Forssmann U, Frimpong-Boateng A, Bals R, Forssmann WG: Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J 2001, 15: 1819-1821.PubMedGoogle Scholar
  10. Liu L, Roberts AA, Ganz T: By IL-1 signaling, monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide. J Immunol 2003, 170: 575-580.View ArticlePubMedGoogle Scholar
  11. Sorensen OE, Cowland JB, Theilgaard-Monch K, Liu L, Ganz T, Borregaard N: Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J Immunol 2003, 170: 5583-5589.View ArticlePubMedGoogle Scholar
  12. Tsutsumi-Ishii Y, Nagaoka I: Modulation of human beta-defensin-2 transcription in pulmonary epithelial cells by lipopolysaccharide-stimulated mononuclear phagocytes via proinflammatory cytokine production. J Immunol 2003, 170: 4226-4236.View ArticlePubMedGoogle Scholar
  13. Yamaguchi Y, Nagase T, Makita R, Fukuhara S, Tomita T, Tominaga T, Kurihara H, Ouchi Y: Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice. J Immunol 2002, 169: 2516-2523.View ArticlePubMedGoogle Scholar
  14. Quinones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J, Rangel HR, Marotta ML, Mirza M, Jiang B, Kiser P, et al.: Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication. AIDS 2003, 17: F39-F48. 10.1097/00002030-200311070-00001View ArticlePubMedGoogle Scholar
  15. Zhang L, Yu W, He T, Yu J, Caffrey RE, Dalmasso EA, Fu S, Pham T, Mei J, Ho JJ, et al.: Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 2002, 298: 995-1000. 10.1126/science.1076185View ArticlePubMedGoogle Scholar
  16. Schaller-Bals S, Schulze A, Bals R: Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med 2002, 165: 992-995.View ArticlePubMedGoogle Scholar
  17. Hiratsuka T, Mukae H, Iiboshi H, Ashitani J, Nabeshima K, Minematsu T, Chino N, Ihi T, Kohno S, Nakazato M: Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis. Thorax 2003, 58: 425-430. 10.1136/thorax.58.5.425PubMed CentralView ArticlePubMedGoogle Scholar
  18. Fang XM, Shu Q, Chen QX, Book M, Sahl HG, Hoeft A, Stuber F: Differential expression of alpha- and beta-defensins in human peripheral blood. Eur J Clin Invest 2003, 33: 82-87. 10.1046/j.1365-2362.2003.01076.xView ArticlePubMedGoogle Scholar
  19. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis Crit Care Med 1992, 20: 864-874.Google Scholar
  20. Sagner G, Goldstein C: Principles, workflows and advantages of the new LightCycler Relative Quantification Software. Biochemica 2001, 3: 15-17.Google Scholar
  21. Docke WD, Hoflich C, Davis KA, Rottgers K, Meisel C, Kiefer P, Weber SU, Hedwig-Geissing M, Kreuzfelder E, Tschentscher P, et al.: Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study. Clin Chem 2005, 51: 2341-2347. 10.1373/clinchem.2005.052639View ArticlePubMedGoogle Scholar
  22. Researcher's toolkit, Statistical Power Calculator, Averages, Two Samples. DSS Research web site[]
  23. Dellinger RP, Carlet JM, Masur H, Gerlach H, Calandra T, Cohen J, Gea-Banacloche J, Keh D, Marshall JC, Parker MM, et al.: Surviving sepsis campaign guidelines for management of severe sepsis and septic shock. Crit Care Med 2004, 32: 858-873. 10.1097/01.CCM.0000117317.18092.E4View ArticlePubMedGoogle Scholar
  24. Boman HG: Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol 1998, 48: 15-25. 10.1046/j.1365-3083.1998.00343.xView ArticlePubMedGoogle Scholar
  25. Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 2002, 415: 389-395. 10.1038/415389aView ArticlePubMedGoogle Scholar
  26. Niyonsaba F, Someya A, Hirata M, Ogawa H, Nagaoka I: Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells. Eur J Immunol 2001, 31: 1066-1075. 10.1002/1521-4141(200104)31:4<1066::AID-IMMU1066>3.0.CO;2-#View ArticlePubMedGoogle Scholar
  27. Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ, et al.: Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 2002, 298: 1025-1029. 10.1126/science.1075565View ArticlePubMedGoogle Scholar
  28. Biragyn A, Belyakov IM, Chow YH, Dimitrov DS, Berzofsky JA, Kwak LW: DNA vaccines encoding human immunodeficiency virus-1 glycoprotein 120 fusions with proinflammatory chemoattractants induce systemic and mucosal immune responses. Blood 2002, 100: 1153-1159. 10.1182/blood-2002-01-0086View ArticlePubMedGoogle Scholar
  29. Ashitani J, Mukae H, Hiratsuka T, Nakazato M, Kumamoto K, Matsukura S: Plasma and BAL fluid concentrations of antimicrobial peptides in patients with Mycobacterium avium-intracellulare infection. Chest 2001, 119: 1131-1137. 10.1378/chest.119.4.1131View ArticlePubMedGoogle Scholar
  30. Beisswenger C, Kandler K, Hess C, Garn H, Felgentreff K, Wegmann M, Renz H, Vogelmeier C, Bals R: Allergic airway inflammation inhibits pulmonary antibacterial host defense. J Immunol 2006, 177: 1833-1837.View ArticlePubMedGoogle Scholar
  31. Dauletbaev N, Gropp R, Frye M, Loitsch S, Wagner TO, Bargon J: Expression of human beta defensin (HBD-1 and HBD-2) MRNA in nasal epithelia of adult cystic fibrosis patients, healthy individuals, and individuals with acute cold. Respiration 2002, 69: 46-51. 10.1159/000049369View ArticlePubMedGoogle Scholar
  32. Schmid M, Fellermann K, Wehkamp J, Herrlinger K, Stange EF: [The Role of defensins in the pathogenesis of chronic-inflammatory bowel disease]. Z Gastroenterol 2004, 42: 333-338. 10.1055/s-2004-813072View ArticlePubMedGoogle Scholar
  33. Smith JJ, Travis SM, Greenberg EP, Welsh MJ: Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 1996, 85: 229-236. 10.1016/S0092-8674(00)81099-5View ArticlePubMedGoogle Scholar
  34. Isomoto H, Mukae H, Ishimoto H, Nishi Y, Wen CY, Wada A, Ohnita K, Hirayama T, Nakazato M, Kohno S: High concentrations of human beta-defensin 2 in gastric juice of patients with helicobacter pylori infection. World J Gastroenterol 2005, 11: 4782-4787.PubMed CentralView ArticlePubMedGoogle Scholar
  35. Mutunga M, Fulton B, Bullock R, Batchelor A, Gascoigne A, Gillespie JI, Baudouin SV: Circulating endothelial cells in patients with septic shock. Am J Respir Crit Care Med 2001, 163: 195-200.View ArticlePubMedGoogle Scholar
  36. Sahly H, Schubert S, Harder J, Kleine M, Sandvang D, Ullmann U, Schroder JM, Podschun R: Activity of human β-defensins 2 and 3 against ESBL-producing klebsiella strains. J Antimicrob Chemother 2006, 57: 562-565. 10.1093/jac/dkl003View ArticlePubMedGoogle Scholar
  37. Singh PK, Tack BF, McCray PB Jr, Welsh MJ: Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol 2000, 279: L799-L805.PubMedGoogle Scholar
  38. Sun L, Finnegan CM, Kish-Catalone T, Blumenthal R, Garzino-Demo P, La Terra Maggiore GM, Berrone S, Kleinman C, Wu Z, Abdelwahab S, et al.: Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection. J Virol 2005, 79: 14318-14329. 10.1128/JVI.79.22.14318-14329.2005PubMed CentralView ArticlePubMedGoogle Scholar
  39. Pitton C, Fitting C, van Deuren M, van der Meer JW, Cavaillon JM: Different regulation of TNF alpha and IL-1ra synthesis in LPS-tolerant human monocytes. Prog Clin Biol Res 1995, 392: 523-528.PubMedGoogle Scholar
  40. Wolk K, Docke W, von Baehr V, Volk H, Sabat R: Comparison of monocyte functions after LPS- or IL-10-induced reorientation: importance in clinical immunoparalysis. Pathobiology 1999, 67: 253-256. 10.1159/000028104View ArticlePubMedGoogle Scholar
  41. Duits LA, Rademaker M, Ravensbergen B, Van Sterkenburg MA, van Strijen E, Hiemstra PS, Nibbering PH: Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids. Biochem Biophys Res Commun 2001, 280: 522-525. 10.1006/bbrc.2000.4157View ArticlePubMedGoogle Scholar
  42. Meyer JE, Harder J, Gorogh T, Weise JB, Schubert S, Janssen D, Maune S: Human beta-defensin-2 in oral cancer with opportunistic Candida infection. Anticancer Res 2004, 24: 1025-1030.PubMedGoogle Scholar
  43. Terai K, Sano Y, Kawasaki S, Endo K, Adachi W, Hiratsuka T, Ihiboshi H, Nakazato M, Kinoshita S: Effects of dexamethasone and cyclosporin A on human beta-defensin in corneal epithelial cells. Exp Eye Res 2004, 79: 175-180. 10.1016/j.exer.2004.03.006View ArticlePubMedGoogle Scholar
  44. Fulop T, Larbi A, Douziech N, Fortin C, Guerard KP, Lesur O, Khalil A, Dupuis G: Signal transduction and functional changes in neutrophils with aging. Aging Cell 2004, 3: 217-226. 10.1111/j.1474-9728.2004.00110.xView ArticlePubMedGoogle Scholar
  45. Lloberas J, Celada A: Effect of aging on macrophage function. Exp Gerontol 2002, 37: 1325-1331. 10.1016/S0531-5565(02)00125-0View ArticlePubMedGoogle Scholar
  46. Plowden J, Renshaw-Hoelscher M, Engleman C, Katz J, Sambhara S: Innate immunity in aging: impact on macrophage function. Aging Cell 2004, 3: 161-167. 10.1111/j.1474-9728.2004.00102.xView ArticlePubMedGoogle Scholar
  47. Zerofsky M, Harel E, Silverman N, Tatar M: Aging of the innate immune response in Drosophila melanogaster . Aging Cell 2005, 4: 103-108. 10.1111/j.1474-9728.2005.00147.xView ArticlePubMedGoogle Scholar


© Book et al.; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.