C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function

Abstract

Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis.

Figure 1

Incubation of neutrophil granulocytes (PMN) with CAAP48 results in cellular activation. PMN activation is shown 30 min after incubation with CAAP48 and the other AAT peptides by changes in the surface expression levels of activation/degranulation markers CD66b, CD63, CD69, and CD62L. Oxidative Burst was determined by detection of intracellular generated reactive oxygen species (oxidation of dihydrorhodamine-123 to rhodamine-123, R-123). Data obtained by using 40 µM and 100 µM peptide concentrations of one exemplary experiment out of four independent experiments are shown. Phorbol myristate acetate (PMA) was used as positive control and 1x PBS diluted in RPMI 1640 (1 : 2) as well as PMN in RPMI 1640 without PBS served as negative control.

Figure 2

The hairpin structure of CAAP48 is responsible for the function of the peptide. For identification of the functional domain of the peptide, we incubated fresh isolated PMN with the hairpin peptide and the sequence until the hairpin (u-hairpin) for 30 min. Cells were analyzed for surface expression of CD66b, CD63, CD69, and CD62L by flow cytometry. The hairpin peptide activates PMN, while the u-hairpin peptide shows no activation. Differential expression of activation markers is shown in histograms of one representative experiment out of four independent experiments using peptide concentrations of 40 µM.

Figure 3

Neutrophil viability in response to CAAP48, CAAP47, scrambled peptide, and VIRIP. After 30 min peptide incubation, PMN (1 × 10⁶ cells/mL) were double stained with Annexin V-FITC and propidium iodide (PI) for 15 min and analyzed by flow cytometry. The percentage of viable cells (PI and Annexin negative) is illustrated for different peptide concentrations. CAAP48 (a) leads to a marked reduction of the viability of neutrophils already at 5 μM (24 ng/μL) while CAAP47 (b) influences viability of PMN not before 40 μM (192 ng/μL). Both the scrambled peptide (c) and VIRIP (d) had no effect. Data of four independent experiments are shown. PMA was used as positive control and 1x PBS diluted in RPMI 1640 (1 : 2) as well as PMN in RPMI 1640 without PBS served as negative control.

Figure 4

Chemotactic response of neutrophils to CAAP48 and the SNP-variant CAAP47. The chemotactic response of isolated neutrophils to CAAP48 and the SNP-variant CAAP47 was examined using disposable Boyden chambers. The number of cells migrating to the lower wells of the chamber was assessed by microscopy and using a hematological analyzer after 60 min. Control: RPMI 1640 medium alone; CAAP48 and CAAP47 were used at a concentration of 100 μM (480 ng/μL). CAAP48/CAAP47 dependent migration was normalized to spontaneous migration in controls. Each bar represents the mean ± SD of three independent experiments.

Figure 5

MIP-1α and IL-8 concentrations after incubation of PMN with AAT peptides. The concentration of MIP-1α and IL-8 in the cell culture supernatant of PMN after peptide stimulation is shown. PMN were incubated with different AAT peptides for two and four hours at concentrations as indicated. MIP-1α and IL-8 were quantified by Bio-Plex. Each bar represents the mean ± SD of three independent experiments. The lower limit of detection for MIP-1α measurements is 1.6 pg/mL and 1.0 pg/mL for IL-8. MIP-1α and IL-8 are released in a dose-dependent manner upon PMN incubation with CAAP47 and CAAP48, while no dose-dependent MIP-1α or IL-8 release could be observed upon stimulation with VIRIP or the scrambled peptide compared to the negative control (1x PBS diluted in RPMI 1640 (1 : 2)). PMA stimulation served as positive control.

Figure 6

Boxplots of CAAP47/48 concentrations. Boxplots of CAAP48 concentrations in severe sepsis and SIRS patients following cardiac surgery and polytrauma are shown. CAAP48 concentrations comprise CAAP48 or CAAP47 concentrations for homozygous individuals and CAAP47/48 concentrations for heterozygous individuals. Circles represent mild outliers and asterisks extreme outliers. p values for significant differences between groups are indicated with respective lines. Severe sepsis patients exhibit significant higher CAAP47/48 concentrations compared to the severe SIRS and HIV infected patients.

Figure 7

Receiver operating characteristic (ROC)-curve analysis comparing the test-characteristics of CAAP47/48 and procalcitonin (PCT) for discrimination of patients with severe sepsis (n = 19) and patients with severe SIRS (polytrauma and cardiac surgery, n = 36). The area under the ROC-curve (AUROC) for CAAP47/48 (0.96) is significantly higher than for PCT (0.62) demonstrating a greater diagnostic accuracy of CAAP47/48 compared to PCT. The optimal cutoff, which is defined to be the concentration at which patients are classified into the SIRS or sepsis group with highest sensitivity and specificity, is 4.9 μM. Severe SIRS and severe sepsis patients can be differentiated with 91.7% specificity and 78.9% sensitivity.

Figure 8

Cleavage sites in the AAT reactive center loop (RCL). Shown are the known cleavage sites in the RCL (italic) of AAT with respective enzymes gathered from the MEROPS database [29]. Human proteases are displayed in black and proteases of other species in grey. Also for every cleavage site the respective molecular weight of the C-terminal peptide is indicated. Cleavage between Phe³⁵² and Leu³⁵³ leads to the generation of CAAP48 (red frame). However in the presence of SNP rs1303 cleavage at this site leads to the generation of CAAP47, a peptide with an E > D substitution at the position indicated in blue.

Figure 9

Hypothetical pathway illustrating CAAP47/48 generation by infection specific proteases. (1) In severe sepsis infection specific proteases are activated that lead to AAT cleavage and thus generation of CAAP47/48 (blue arrows). This proteolytic fragment activates PMN, which in turn leads to secretion of further proteases from PMN that will result in additional cleavage of ATT and accumulation of CAAP47/48 (2), that subsequently activates other PMN (red arrows). Due to the activation of further PMN and the induction of migration, cell death, and oxidative burst, this activation loop contributes to a strong enhancement of inflammation.