Early immune anergy towards recall antigens and mitogens in patients at onset of septic shock

Abstract

The pathology of sepsis is typically characterized by an infection and excessive initial inflammation including a cytokine storm, followed by a state of immune suppression or paralysis. This classical view of a two peak kinetic immune response is currently controversially discussed. This study was a sub-study of the randomized clinical Trial SISPCT registered with www.clinicaltrials.gov (NCT00832039, Registration date: 29/01/2009). Blood samples from 76 patients with severe sepsis and septic shock were incubated for 48 h at 37 °C in vitro with bacterial or fungal recall-antigens or specific mitogen antigens within 24 hours of sepsis onset. Recall-antigen stimulation led to a severe dampening of normal cytokine release. This immunologic anergy was similarly observed after mitogen stimulation. Moreover, patients under hydrocortisone therapy or with lowered arterial oxygen tension had further reductions in cytokine levels upon B- and T-cell mitogen stimulation. This investigation reveals an early onset of immunoparalysis during sepsis. This immune incompetence in mounting an adequate response to further infections includes previously sensitized pathogens, as seen with recall-antigens. Also, the immune-suppressive role of hydrocortisone and low PaO2 is highlighted. Aside from early broad-spectrum antimicrobial therapy, our findings reinforce the need for maximal immunological support and protection against further infections at the onset of sepsis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Patients versus control group in unstimulated assay and after stimulation with recall antigens. SS: severe sepsis/septic shock patients, Basal: unstimulated test assay, Bacteria: bacterial antigen mixture, Fungi: fungal antigen mixture. Blood samples were taken subsequently to study enrolment (SS) or at a time of subjective physical well-being (control group), respectively. In boxplots, boxes show the median and interquartile range (IQR), whiskers represent the 10th and 90th percentile. Statistically significant differences (Mann-Whitney Rank Sum Test) are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. y-axis: logarithmic scale.

Figure 2

Patients versus control group after stimulation with PWM, PMA, CD3/28 and LPS. PWM: Pokeweed mitogen, LPS: Lipopolysaccharide, PMA-I: Phorbol myristate acetate and Ionomycin, CD3/28: Cluster of Differentiation 3/28, SS: severe sepsis/septic shock patients. Blood samples were taken subsequently to study enrolment (SS) or at a time of subjective physical well-being (control group), respectively. In boxplots, boxes show the median and interquartile range (IQR), whiskers represent the 10th and 90th percentile. Statistically significant differences (Mann-Whitney Rank Sum Test) are indicated as follows: *p 

Figure 3

Relationship between disease severity and TNF-α release in Bacteria and PWM assay. TNF-α release from whole blood was correlated with quartile groups for disease severity as measured by SAPS II (A,C) and APACHE II (B,D) disease severity classification systems. Panels A and B show hereby stimulations with bacterial antigen mixtures, Panels C and D with Pokeweed mitogen assay. Statistically significant differences (One-way ANOVA on RANKS followed by Dunn’s test) are indicated *p < 0.05.

Figure 4

Relationship between hydrocortisone administration and cytokine release. Patients were allocated to two groups (Hydrocortisone (HC) or no HC) for comparison of stimulated cytokine release. (A) TNF-α in supernatants of whole blood stimulated with PWM. (B) IL-1β in supernatants of LPS stimulated whole blood. Statistically significant differences (Mann-Whitney Rank Sum Test) are indicated as follows: **p < 0.01, ***p < 0.001.

Figure 5

Relationship between PaO2 and cytokine release. Patients were allocated to groups regarding their arterial partial pressure of oxygen (PaO2) for comparison of stimulated cytokine release: hypoxemia (PaO2 < 80 mmHg) and normoxemia (PaO280–100 mmHg). (A) TNF-α measured after stimulation with PWM. (B) IL-1β measured after stimulation with LPS. Statistically significant differences (Mann-Whitney Rank Sum Test) are indicated *p < 0.05.

Figure 6

ROC curves for the endpoint‘‘RRT during ICU stay” ROC-curves for (A) whole blood response, (B) established parameters used in sepsis and (C) different combinations of markers referring to (A and B) regarding the endpoint “initiation of RRT during ICU stay”: ROC: Receiver operating characteristic curve, all markers combined: combination of PWM stimulated TNF-α release, LPS stimulated IL-1β release, SAPS II, APACHE II and serum IL-6.