The neutrophil‐activating proteins interleukin 8 and β‐thromboglobulin: in vitro and in vivo comparison of NH₂‐terminally processed forms

Abstract

Isolation of the human neutrophil activating protein (NAP) interleukin 8 (IL8) from leukocytes has revealed that it is structurally related to beta-thromboglobulin (beta TG) from platelets. Both these proteins occur as natural mixtures of multiple forms, differing from each other by unequal truncation at the NH2 terminus. In this study we have compared IL8 and beta TG forms for in vitro and in vivo neutrophil activation. In contrast to IL8, none of the beta TG forms were found to exert granulocyte chemotactic activity in vitro, as measured in the agarose assay. However, fractions rich in the most extensively processed forms of beta TG (e.g. NAP-2) as well as pure NAP-2 did induce lactoferrin release from granulocytes, whereas fractions containing only the longer forms (e.g. connective tissue-activating peptide III) were inactive. In order to observe this in vitro effect, about 10-fold less IL8 (10 nM) than NAP-2 was required. In the presence of a vasodilator substance low doses (2-20 pmol) of IL8 and the shorter forms of beta TG caused granulocyte accumulation and plasma leakage in rabbit skin whereas the longer forms of beta TG again failed to do so. Finally, granulocytosis induction following i.v. injection was found to occur with NAP-2. At the maximal dose tested (250 pmol), this in vivo effect of NAP-2 was less pronounced than that of IL8. In the case of IL8, NH2-terminal processing did not seem to affect granulocyte stimulatory activity. It should be noted, however, that the extent of processing of IL8 is less than that occurring with beta TG. It can be concluded that the platelet factor beta TG, structurally related to the monokine IL8, can also play a role in neutrophil activation during inflammatory reactions.

Bacterial-induced release of inflammatory mediators by bronchial epithelial cells

“…More recent findings from our laboratory have indicated that the H. influenzae endotoxin-induced increase in neutrophil chemotaxis can also be blocked by the addition of IL-8 monoclonal neutralizing antibody to the conditioned medium, and suggest that IL-8 is most likely to be involved in this process. This is in agreement with the findings of others, who have also demonstrated that this cytokine primarily influences neutrophil activation [84]. Although others have also demonstrated that epithelial cell derived mediators are chemotactic towards neutrophils [85,86], and indeed other cell types including lymphocytes [87,88] and monocytes [86,89], in vitro, the majority of these studies have employed animal models.…”

Connective tissue activation. xxxv. detection of connective tissue activating peptide–iii isoforms in synovium from osteoarthritis and rheumatoid arthritis patients: patterns of interaction with other synovial cytokines in cell culture

“…Both native and chemically synthesized NAP-2 have been shown to induce cytosolic free calcium, elastase release, and chemotaxis in human neutrophils (40,41). Other data indicate that CTAP-I11 is not an attractant; chemotactic activity appears to result from amino terminal cleavage to the CTAP-111 des 1-13 and des 1-15NAP-2 isoforms, which are able to interact with the NAP-I/ IL-8 receptor on human neutrophils (42).…”

Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties

“…For example, limited NH 2 -terminal truncation of IL-8 (eg, by gelatinase B) enhanced its chemotactic potency, whereas platelet basic protein was found to become chemotactic only when truncated into NAP-2. 21,40 In contrast, small modifications of the NH 2 -terminal residues of MCP-1, MCP-2, and MCP-3 decreased the biologic activity of these CC chemokines. 22,23 Recently, the highly specific protease CD26/DPP IV has been reported to process a number of chemokines at the NH 2 terminus with different effects on their chemotactic and antiviral activities.…”

“…Natural IL-8 was purified to homogeneity from conditioned medium of monocytes and contained an equimolar mixture of IL-8(1-77), IL-8(5-77) and IL-8(6-77). 21 Soluble natural human CD26/DPP IV, without membrane anchor and starting at amino acid Gly31, was obtained from total seminal plasma and purified to homogeneity by anion exchange followed by affinity chromatography on immobilized adenosine deaminase as described. 30 Human CXCR3 was stably expressed in Chinese hamster ovary K1 (CHO-K1) cells (American Type Culture Collection [ATCC], Manassas, VA: CCL-61) and the cells were cultured in HAM F-12 medium (Biowhittaker Europe, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 400 g/mL geneticin.…”

“…Some chemokines become chemotactic only when processed at the NH 2 -terminus, for example, truncation of platelet basic protein (PBP) into NAP-2 (CXCL7). 21 Others, for example, the monocyte chemotactic proteins MCP-1 (CCL2), MCP-2 (CCL8), and MCP-3 (CCL7), lose their chemotactic activity when NH 2 -terminal amino acids are cleaved off. 22,23 Aminopeptidases or endopeptidases that process chemokines at the NH 2 -terminus play an important role in the up-regulation or down-regulation of chemokine activities.…”