(Received for publication, August 16, 1995; and in revised form, November 30, 1995)
From the
Reports that interleukin-8 (IL-8) induces the infiltration of
neutrophils followed by T-cells into injection sites led us to
postulate that by stimulation of neutrophil degranulation IL-8 may
cause the release of factors with chemoattractant activity for
T-lymphocytes. Extracts of human neutrophil granules were
chromatographed to isolate and purify T-lymphocyte chemoattractant
factors. Two major peaks of T-cell chemotactic activity were purified
by C18 reversed phase high pressure liquid chromatography (HPLC). The
first peak was resolved further by C4 reversed phase HPLC and yielded
an active fraction shown by NH-terminal amino acid sequence
analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified
defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA)
were also potent chemoattractants for human T-cells, while HNP-3 was
inactive. The second peak of T-cell chemoattractant activity was also
further purified to homogeneity by C4 reversed phase HPLC and
identified by NH
-terminal sequence analysis as
CAP37/azurocidin, a protein with sequence homology to serine proteases.
0.1-100 ng of defensins and 1.0-100 ng/ml CAP37 were able
to stimulate in vitro T-cell chemotaxis. Neutrophil activating
factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin,
and formylmethionylleucylphenylalanine each induced the release of
CAP37 and defensins from neutrophil granules. Subcutaneous
administration of defensins or CAP37/azurocidin into BALB/c mice
resulted in a moderate neutrophil and mononuclear cell infiltrate by 4
h, which was greater by 24 h at the site of injection. Additionally,
subcutaneous injection of defensins into chimeric huPBL-SCID mice
resulted in significant infiltration by human CD3+ cells within 4
h. These results identify the antimicrobial proteins, CAP37/azurocidin
and defensins HNP-1 and HNP-2, as potent neutrophil-derived
chemoattractants for T-cells. These proteins represent primordial
antimicrobial peptides which may have evolved into acute inflammatory
cell-derived signals that mobilize immunocompetent T-cells and other
inflammatory cells.
Immunologically induced inflammatory responses are multistep
processes involving the production of various chemotactic factors
resulting in the orchestrated recruitment of neutrophils, mast cells,
monocytes, and T-cells. The chemokine(1, 2) IL-8, ()a potent chemoattractant and activator of neutrophils, is
produced by many cell types including
neutrophils(3, 4) , monocytes(5, 6) ,
eosinophils(7) , and endothelial cells(8) . Two
receptors for IL-8 have been cloned and belong to the family of seven
transmembrane G protein-coupled receptors(9, 10) .
Signaling through these receptors induces a number of biochemical and
biological events including the mobilization of intracellular calcium (11) , changes in adhesion(12) , respiratory
burst(13) , degranulation, and enzyme release from
neutrophils(6, 11, 14, 15) .
IL-8 has also been reported to be an in vitro and in vivo chemoattractant for T-cells(16) . Furthermore, we have demonstrated that subcutaneous injection of human IL-8 into human T-cell-engrafted SCID mice causes an initial infiltration of murine neutrophils by 4 h into the site of injection, which is followed by a marked infiltration of human T-cells by 72 h(17) . This delayed infiltration of human T-cells suggested that IL-8 may recruit T-cells indirectly, perhaps by stimulating the release of T-cell chemotactic factors by activated neutrophils. This hypothesis was tested by incubating purified peripheral blood neutrophils with a number of degranulating agents, including IL-8, and resulted in the release of T-cell chemotactic activity into the supernatants within 2 h. This led us to detect, purify, and identify several T-cell chemotactic factors from extracts of neutrophil granules.
Figure 1: Partial purification of neutrophil granule-derived T-cell chemotactic factors by reversed phase HPLC. Acid- and salt-extracted proteins from neutrophil granules were loaded on a C18 Radial-Pak HPLC column and eluted with a linear gradient of acetonitrile(- - -). The absorbance was monitored at 206 nm. Protein-containing fractions were tested for T-cell chemoattractant activity using the microchamber assay. hpf, high power field.
Figure 2: Purification of defensins by C4 reversed phase chromatography. The first peak of activity from the C18 column was lyophilized, loaded onto a C4 reversed phase HPLC column and eluted with a gradient of acetonitrile(- - -). Fractions were tested for activity using the microchamber assay. The active fraction, indicated by an open rectangle beneath the absorbance trace, showing one peptide band on gradient (10-20%) Tris/Tricine gel with Coomassie staining (see inset) was sequenced and identified as HNP-1, HNP-2, and HNP-3.
Figure 3: Purified defensins and CAP37/azurocidin are chemotactic for T-cells. C4 purified defensin, (a mixture of HNP-1, HNP-2, and HNP-3) and CAP37/azurocidin were tested at different concentrations for T-cell chemotactic activity. MIP-1b (5 ng/ml) is included as a positive control. The migration was performed using the Boyden chamber assay on collagen-coated filters as described under ``Materials and Methods.'' The results in this representative experiment are expressed as the mean number of T-cells that have migrated through the filter per high power field.
The T-cell chemotactic activity of homogeneous preparations of neutrophil-derived HNP-1, HNP-2 and HNP-3 (kindly provided by Dr. R. I. Lehrer, UCLA) were compared with our preparation. The dose response to individual HNP-1 and HNP-2 was practically identical to that of our preparation containing a mixture of HNP-1, HNP-2, and HNP-3 (Fig. 3). Checkerboard analysis of our HNP-1 and HNP-2 showed that their effect was chemotactic rather than chemokinetic (data not shown). HNP-3, which differs from HNP-1 only by substitution of aspartate for the amino-terminal alanine, was not able to stimulate T-cell chemotaxis. It has been proposed that HNP-3 is a precursor of HNP-2 (24) and proteolytic removal of the amino-terminal aspartate residue converts the inactive HNP-3 into chemotactically active HNP-2 (24) . While serum proteins have been reported to inhibit the antimicrobial activity of defensins(24) , both HNP-1 and HNP-2 retained in vitro chemotactic activity for human T-cells in the presence of 10% human serum (data not shown).
Figure 4: Purification of CAP37/azurocidin by C4 reversed phase chromatography. The second peak of activity from the C18 column was lyophilized, loaded onto a C4 reversed phase HPLC column and eluted with a gradient of acetonitrile(- - -). Fractions were tested for activity using the microchamber assay. The active fraction, indicated by the open rectangle and showing one protein band on 15% Tris/glycine SDS-PAGE, was sequenced and identified as CAP37/azurocidin.
Figure 5:
IL-8, FMLP, and PMA/ionomycin stimulate
the release of defensins from neutrophils in vitro. Human
neutrophils were cultured with stimulant for 4 h at 37°C after
which supernatants were collected and concentrated. The concentrated
supernatant was subjected to SDS-PAGE on 4-20% Tris/glycine gel
followed by Western blot analysis with anti-defensin rabbit antisera.
The stimulants were: lane 1, media control; lane 2,
IL-8 (50 ng/ml); lane 3, IL-8 plus cytochalasin B (5
µg/ml); lane 4, FMLP (10M); lane 5, FMLP plus cytochalasin B; lane 6, PMA (2
ng/ml) plus ionomycin (500 nM); lane 7, 1 µg of
purified defensin preparation.
The results presented in this report demonstrate that
neutrophils are an important source of factors chemotactic for
T-lymphocytes. The polypeptides derived from neutrophil granules were
identified as T-cell chemotactic factors: defensins NP-1 and -2 and
CAP37/azurocidin. Defensins are a family of small (29-30 amino
acids, approximately 3.5 kDa) cationic antimicrobial proteins (24) whose structure is stabilized by three conserved disulfide
bridges, including one disulfide that cyclizes the protein by linking
the carboxyl-terminal cysteine to the amino-terminal ultimate (HNP-2)
or penultimate (HNP-1, HNP-3) cysteine (29) . Indeed the
defensins HNP-1, HNP-2 and HNP-3 differ by only a single
NH-terminal amino acid. Approximately 25-30% of the
human azurophilic granule protein consists of defensins. It has been
estimated that up to 3-5 pg of defensins can be released by each
neutrophil. They are believed to be released into the phagocytic
vesicle of the neutrophil and into the medium (30) where they
contribute to the respiratory burst-independent cytotoxic killing of
microbes. The cytotoxic effect of defensins occurs at micromolar
concentrations and is thought to be due to their ability to form
numerous transmembrane channels that permeabilize lipid bilayers of
microorganisms. Even though their amino acid sequences are virtually
identical, HNP-3 does not kill Candida albicans(31) whereas HNP-1 and HNP-2 have been reported to have
this activity. HNP-3 also lacked T-cell chemotactic activity, but
cleavage of the amino-terminal aspartate residue can convert it into
active HNP-2.
CAP37/azurocidin, like defensins, was previously identified as a neutrophil granule protein also with antimicrobial activity at micromolar concentrations(32) . Structurally, CAP37 belongs to the serine protease superfamily and has 45% sequence identity with human neutrophil elastase; however, due to replacements of crucial amino acids at the active site, CAP37/azurocidin is inactive as a protease (26) . It is unlikely that there are any other T-cell chemotactic peptides present that we have not detected in the neutrophil granule extracts because the defensins and CAP37 are major protein constituents of neutrophil granules and are chemotactic at low (nanomolar) concentrations.
Both the defensins HNP-1 and HNP-2 and
CAP37/azurocidin have been reported to chemoattract
monocytes(35, 36) . However, we have repeatedly been
unable to detect any monocyte or neutrophil chemotaxis with doses of
1-1000 ng/ml defensins. The report that defensins chemoattract
human monocytes is based on assays of the leading front of mononuclear
cells migrating into a filter(35) . It is difficult to identify
cells lodging in a filter, and this may have led to a misidentification
of the migrating mononuclear cell type. Our assay method more readily
permits the identification of cells adhering to the underside of
polycarbonate filters as T-cells. In the case of our preparations of
natural CAP37/azurocidin and the purified and recombinant CAP37
(provided us by Dr. A. Pereira), we also could not detect significant
monocyte chemotaxis (chemotaxis index 1.5). Perhaps this is based
on technical discrepancies in our assays, or possibly tissue
macrophages express more receptors for CAP37 than do human peripheral
monocytes. However, we have identified another protein present in the
chromatography fractions of the neutrophil granule extracts as having
monocyte chemoattractant activity but not T-cell chemotactic activity.
This protein migrates very closely to CAP37/azurocidin and may
therefore have been a contaminant of CAP37 preparations that attract
monocytes. (
)Our data indicate that defensins and
CAP37/azurocidin are potent in vitro T-cell chemoattractants.
Because defensins account for 25-30% of the neutrophil granule protein content, they may serve as a major source of T-cell chemoattractant whose release is under the control of neutrophil activators such as IL-8. Although serum proteins effectively inactivate the antimicrobial activity of defensins, their in vitro chemotactic activity is not impaired by the presence of 10% fetal calf serum (data not shown). This predicts that defensins can deliver a T-lymphocyte activating signal at a distance from the site of acute inflammation where they originate. This prediction was borne out by our experiments showing that BALB/c mice injected subcutaneously with 1.0-µg injections of defensins or CAP37/azurocidin developed mononuclear as well as neutrophilic infiltrates by 4 h. By 24 h the number of accumulated neutrophils and mononuclear cells was markedly increased even in response to lower 10- or 100-ng doses. The infiltration by neutrophils and mononuclear cells was not predicted by the in vitro assays. It is possible that defensins and CAP37/azurocidin initiate a cascade of proinflammatory signals in vivo resulting in the recruitment of neutrophils and monocytes. The inflammatory response may be a result of the tissue-damaging effects of these microbicidal agents, although necrosis became evident only at 24 h in response to the 1000-ng dose. In the case of the defensins, immunohistochemical studies revealed that in four of the six chimeric huPBL-SCID mice, CD3+ human T-cells appeared at the injection site already by 4 h. However, there are also unstained infiltrating mononuclear cells which are murine macrophages. These macrophages may express more receptors for human defensins and CAP37/azurocidin. The possibility that these agents are immunomodulators and may have adjuvant activity is currently being tested.
Our quantitative assays of the Western blots revealed that
IL-8, FMLP, and phorbol 12-myristate 13-acetate/ionomycin resulted in
comparable release of defensins by neutrophils in vitro. On
the presumption that IL-8 induces similar degranulating effects in
vivo, we therefore estimate that 2 10
neutrophils could release 600 ng of defensins in response to
IL-8. This would produce a concentration gradient sufficient to attract
T-lymphocytes. Preincubation of neutrophils with tumor necrosis
factor-
has also been shown to augment the IL-8-stimulated
degranulation of both azurophilic and specific granules of
neutrophils(27) , suggesting that secondary inflammatory
stimuli may have a priming effect on IL-8-stimulated degranulation and
can thus enhance leukocyte recruitment to sites of inflammation.
The physiological importance of these neutrophil granule T-lymphocyte chemoattractants remains to be established. Tantalizing clues are provided by an experiment of nature namely beige mutant mice with the Chédiak-SteinbrinckHigashi Syndrome. Beige mice have also been reported to be deficient in defensins(37, 38) . An even less common disorder, specific granule deficiency, is also characterized by a deficiency in defensins and increased susceptibility to microbial infections(39) . The defective host defenses in these mice may be due in part to deficient secretion of defensins and CAP37/azurocidin. Our results indicate that the release of these proteins from neutrophils can be triggered by proinflammatory mediators, such as IL-8, in addition to the release caused by phagocytosis of microorganisms(30, 35) . The possibility that these potent T-lymphocyte chemoattractants can provide a means by which acute inflammatory reactions develop into immunologically mediated chronic inflammatory responses dominated by mononuclear cells is supported by data showing that depletion of neutrophils inhibits subsequent chronic inflammatory reactions in mice (17) .