Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1

Abstract

Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits.

Fig 1. fMYFINILTL and MYFINILTL induced superoxide production from human neutrophils.

Neutrophil NADPH-oxidase activity was measured by an isoluminol/HPR chemiluminescence system. (A) Neutrophils were pre-incubated at 37°C for 5 min in the absence (solid line) or presence of FPR1 antagonist Cyclosporin H (CysH, 1 μM, dashed line) or FPR2 antagonist PBP₁₀ (1 μM, dotted line) before stimulation with fMYFINILTL (10 nM; time for addition indicated by an arrow). The curves are from one representative response (n > 10). Inset: The superoxide production induced by the non-formylated peptide variant MYFINILTL (1 μM) in the presence or absence (control) of FPR specific inhibitors. Abscissa, Time of study (min); ordinate, superoxide production (in arbitrary light units, cpmx10^-6). (B) Neutrophil NADPH-oxidase activity induced by different concentrations of fMYFINILTL. The peak values of the responses in relation to the concentration of fMYFINILTL was determined and is expressed as a percent of the maximal response. Data are expressed as mean ± SD from four independent experiments. Abscissa, agonist concentration (M); ordinate, superoxide production (percent of max).

Fig 2. fMYFINILTL induced superoxide production from bone marrow derived mouse neutrophils.

Neutrophil NADPH-oxidase activity was measured by an isoluminol/HPR chemiluminescence system. (A) Neutrophils (5x10⁴) were activated by either fMYFINILTL (50 nM) or fMIFL (10 nM). The time point for addition of agonists is indicated by an arrow and the curves are from one representative response (n > 3). Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6). (B) Mouse neutrophil NADPH-oxidase activity induced by different concentrations of fMYFINILTL. The peak values of the responses in relation to the concentration of fMYFINILTL was determined and is expressed as a percent of the maximal response. Data are expressed as mean ± SD from three independent experiments. Abscissa, agonist concentration (M); ordinate, superoxide production (percent of max).

Fig 3. NADPH-oxidase activity induced in bone marrow neutrophils derived from wild-type and Fpr2^-/- mice by fMIFL and PSMα2.

The oxidase activity was measured by an isoluminol/HPR chemiluminescence system. (A) Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 10 nM fMIFL. (B) Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 50 nM PSMα2. (C) Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 50 nM fMYFINILTL. Arrows indicate the time points for agonist addition and a representative experiment out of four is shown. Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6).

Fig 4. NADPH-oxidase activity induced in mouse (A) and human (B) neutrophils by formyl peptides differing in their M3 binding properties, fMWYYLF (a representative of the low M3 binding group), fMFLIDV (a representative of the middle M3 binding group) and, fMILLV (a representative of the low M3 binding group).

(A) Neutrophils (5x10⁴) from wild type (WT; solid lines) and Fpr2^-/- (broken lines) were activated by by fMWYYLF (5 nM), fMFLIDV (5 nM) or fMILLV (5 nM) and the oxidase activity was measured. The time point for addition of the agonist is indicated by an arrow. B) Human neutrophils incubated 5 min without (solid lines) or with an FPR antagonist, either the FPR2 antagonist PBP₁₀ (1 μM, dashed lines) or the FPR1 antagonist CysH (1 μM, dotted lines) were activated with fMWYYLF (100 nM), fMFLIDV (500 nM) or fMILLV (5 nM) and the oxidase activity was measured. The time point for addition of the agonist is indicated by an arrow. Arrows indicate the time points for addition of the peptide agonists. One representive experiment out of three is shown. Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6).

Fig 5. The fmYFINILTL peptide activates both human and mouse neutrophils.

A. Dose response of the superoxide release induced by fmYFINILTL from human neutrophils. The peak values of the responses in relation to different concentrations of fmYFINILTL were determined and are expressed as percent of the maximal response. Data are expressed as mean ± SD from three independent experiments. Inset: Human neutrophils were pre-incubated in the absence (solid line) or presence of FPR2 antagonist PBP₁₀ (1 μM, dashed line), or FPR1 antagonist CysH (1 μM, dotted line) followed by stimulation with fmYFINILTL (250 nM). An arrow indicates the time for addition of the agonist. Representive responses from one experiment out of three are shown. Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6). as indicated by the arrow. The figures represent one out of three experiments. B. The fmYFINILTL peptide (2 μM) induced superoxide production from wild type or Fpr2^-/- neutrophils. Data are expressed as mean ± SD of the peak response of fmYFINILTL in percent of the peak fMIFL response (n = 3).