Neutrophil-activating factor

Details Neutrophil-activating factor Neutrophil-activating factor

1995-06-06

2002-05-07

Mertz, Prema (Department: 1646)

Drug, bio-affecting and body treating compositions

Lymphokine

C530S324000, C530S344000, C514S002600, C514S885000

Reexamination Certificate

active

06383479

ABSTRACT:

The present invention is concerned with an immunomodulatory substance.
It is more particularly concerned with an immunostimulating factor capable of activating human neutrophil leukocytes, hereinafter designated neutrophil-activating factor (NAF).
1. BACKGROUND
The neutrophil leukocytes (neutrophils) are the most common leukocytes and account for about ⅔ of the white cells in human blood. They have one main function, which is to protect the host organism against microbial infections. The neutrophils are mobile, are responsive to chemotactic stimuli generated upon infection, and are able to move into infected tissues to kill the microorganisms. The killing depends on the ability of the neutrophils to engulf the microorganisms and to release oxygen radicals and microbiocidal enzymes (B. M. Babior,
New Engl. J. Med.
298 [1978] 659-668 and M. Baggiolini,
Experientia
40 [1984 ] 906-909). The release of such products depends on activation of the neutrophils. Substances like the neutrophil-activating factor described herein could thus be employed to enhance neutrophil activation and therefore the antimicrobial defense mechanism of the human body.
We have now found that human monocytes secrete a factor that induces exocytosis and the respiratory burst (oxygen radical formation and enzyme release) in human neutrophils and thus displays neutrophil-activating properties which are important in antimicrobial activity.
The factor can be obtained from the culture fluid of stimulated human monocytes and has an apparent molecular weight of approximately 6000, more precisely about 6500, in SDS-gel electrophoresis.
Monocytes are phagocytic cells similar to neutrophils. In contrast to neutrophils, however, monocytes are long-lived. They migrate from the blood into all possible tissue sites where they transform into macrophages. The transformation from monocytes to macrophages can also be observed in cell culture experiments. The macrophages in the tissues have a variety of functions, mainly phagocytosis of unwanted material and production of a great variety of peptides and proteins which are secreted. Macrophages collect at the site of persisting infection and it is at these locations that the production of NAF by these cells could enhance the host-defence capabilities of neutrophils and thus have a pathophysiologically relevant function.
NAF is characterized by its neutrophil-activating properties, i.e. the induction of the so-called respiratory burst with production of oxygen radicals and the induction of enzyme release. In molecular terms NAF, is characterized by the following properties: an apparent molecular weight of about 6500 in SDS-gel electrophoresis, an isoelectric point of approximately 8.6, resistance to heat up to 80° C. and to a number of denaturing agents but susceptibility to proteases, suggesting that NAF is a polypeptide. The action of NAF on human neutrophils is similar to that of two known chemotactic stimuli, the anaphylatoxin C5a and the bacterial peptide N-formyl-L-methionyl-L-leucyl-L-phenyl-alanine (fMLP), but is mediated by a surface receptor to which NAF binds and which differs from the receptors of any known agonist of human neutrophils. NAF is produced in culture by human monocytes but not by human lymphocytes. Production depends on the presence of a stimulus like bacterial lipopolysaccharide (LPS), phytohaemagglutinin (PHA), or concanavalin A (ConA) and on stimulus concentration and incubation time. It is inhibited by cycloheximide, indicating that de novo synthesis of protein is involved.
As already indicated, monocytes and macrophages are abundant sources of a variety of bioactive peptides and proteins (C. F. Nathan,
J. Clin. Invest.
79 [1987] 319-326). They have been identified as the producers of three distinct cytokines: interleukin l (IL-l), tumor necrosis factor (TNF) and interferon-alpha (IFN-&agr;). A number of reports have been published showing that monocytes and macrophages also produce factors acting on neutrophils which are different from those mentioned above. Since they are active on neutrophils these factors are presented hereafter in some detail. It was reported in various publications that alveolar macrophages release factors which are chemotactic for neutrophils (J. A. Kazmierowski et al.,
J. Clin. Invest.
59 [1977] 273-281; W. W. Merrill et al.,
J. Clin. Invest.
65 [1980] 268-270 and G. W. Hunninghake et al.,
J. Clin. Invest.
66 [1980] 473-483) and which can enhance the antimicrobial defense in the lung. It was shown subsequently that these factors enhance the microbiocidal activity of neutrophils (J. E. Pennington et al.,
J. Infect. Dis.
148 [1983] 101-109 and
J. Clin. Invest.
75 [1985] 1230-1237). Purification by gel filtration and chromatofocusing led to the identification of a protease-sensitive factor (termed NAF) produced by alveolar macrophages, with a molecular weight of 6000 and an isoelectric point of 7.6. This factor was reported to be weakly chemotactic and to enhance the killing of phagocytosed bacteria by neutrophils without, however, inducing by itself the production of oxygen radicals nor the release of enzymes. A similar mechanism of enhanced anti-microbial activity was described by other workers (A. Ferrante et al.,
Clin. Exp. Immunol.
56 [1984] 559-566), who showed that human neutrophils required the addition of culture media from monocytes or macrophages to induce the killing of
Neigleria fowleri
. Granulocyte-activating mediators (GRAM) produced by LPS-simulated human monocytes were described by other laboratories (A. Kapp et al.,
J. Invest. Dermatol.
86 [1986] 523-528 and F. E. Maly et al.,
Lymphokine Res.
5 [1986] 21-33). Two GRAM species were described, a major one with an apparent molecular weight of 60,000 and a minor one with an apparent molecular weight of 10,000, which induced a delayed respiratory burst response in human neutrophils, as revealed by chemiluminescence. These factors are sensitive to heat and trypsin and their production is dependent on stimulation of the monocytes with LPS and, apparently, on de novo protein synthesis.
Several reports describe factors derived from monocytes and/or macrophages with chemotactic activity towards neutrophils. A factor termed “mononuclear cell-derived chemotaxin” (MOC), apparently a peptide with molecular weight of 10,000 which differs from GRAM was reported, (E. Kownatzki et al.,
Clin. Exp. Immunol.
64 [1986] 214-222). A neutrophil chemotactic factor produced by human blood monocytes stimulated with LPS, with a molecular weight of approximately 10,000 and an isoelectric point of 8-8.5 was also known (T. Yoshimura et al.,
J. Immunol.
139 [1987] 788-793). Finally it was also reported that rat peritoneal macrophages stimulated in culture with LPS release a selective neutrophil chemotactic factor (F. Q. Cunha et al.,
J. Med. Biol. Res.
19 [1986] 775-777 and
Eur. J. Pharmacol.
129 [1986] 65-76).
Due to the preliminary nature of the biochemical information contained in these reports it is impossible to speculate about structural similarities and differences among the various factors described.
Subsequently to the priority date(s) which is (are) being claimed for the present invention, purification of a peptide probably corresponding to NAF was described by van Damme et al. (J. Van Damme et al.,
J. Exp. Med.
167 [1988] 1364-1376) and a sequence identical to that of NAF was reported by Gregory et al., (H. Gregory et al.,
Biochem. Biophys. Res. Commun.
151 [1988] 893-890) for a peptide that was purified from supernatants of lectin-stimulated human lymphocytes. The cDNA coding for MDNCF (T. Yoshimura et al.,
Proc. Nat. Acad. Sci. USA
84 [1987] 9233-9237) was recently cloned (K. Matsushima et al.,
J. Exp. Med.
167 [1988] 1883-1893) and found to correspond to the 3-lOC cDNA (J. Schmid and C. Weissmann,
J. Immunol.
139 [1987] 250-256). Schm

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