Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is HLeu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-HisLeu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing ␤-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G i2 -type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce ␤-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.Neutrophils are a type of leukocyte involved in the innate defense system. Once tissue injury occurs because of an infection or toxic cell debris resulting from cell necrosis, these cells migrate from the bloodstream to the injury sites. They are then activated to produce superoxide and digestive enzymes and to phagocytose toxic cell debris and the infectious microorganisms (1, 2). Chemokines, such as interleukin 8, are produced at tissue injury sites where they induce the migration and activation of neutrophils (3). Most of these chemokines are synthesized after the inflammatory stimuli, suggesting that they might not be the substances promoting the initial migration and activation of neutrophils because the transmigration of these cells is often observed immediately after tissue injury. Thus, it is likely that there are neutrophil-activating substances inducing the initial phase of migration. Some of these substances may be present in mitochondria because the contents of disrupted mitochondria, which are thought to be released immediately after necrosis in damaged tissues, promote neutrophilic migration (4). The responsible proteins or peptides, however, have not yet been purified. In the present study, we purified and identified a novel class of neutrophil-activating peptides from healthy porcine hearts that activate primary human neutrophils and neutrophilic/granulocytic differentiated HL-60 cells (HL-60-derived neutrophilic/granulocytic cells). 5 EXPERIMENTAL PROCEDURESPeptides that activated HL-60-derived neutrophilic/granulocytic cells were purified from healthy porcine hearts (outlined in Fig. 1) by monitoring the activity of fractions that induce ␤-hexosaminidase release from the cells. This enzyme can be easily assayed and is known to be quantitatively released from * This study was supported by a re...

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide “mitocryptide-2” (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced β-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of β-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca2+ concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of Gi- or Go-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated β-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [3H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on β-hexosaminidase (β-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced β-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

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