Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes

E. Andersson1, O.E. Sørensen3, B. Frohm1, N. Borregaard3, A. Egesten2 and J. Malm1,4

1 Department of Laboratory Medicine, Divisions of Clinical Chemistry and 2 Medical Microbiology, Lund University, University Hospital MAS, SE- 205 02 Malmö, Sweden and 3 Granulocyte Research Laboratory, Department of Hematology, University Hospital, DK-2100 Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. METHODS AND RESULTS: In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. CONCLUSIONS: These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.

Key words: cathelicidin/hCAP-18/LL-37/prostasome/semen


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Innate immunity is important for the defence of the body against potentially invasive pathogenic micro-organisms. In contrast to adaptive immunity, it provides a rapid and non-specific response (Fearon and Locksley, 1996Go). In recent years, several antimicrobial peptides have been discovered to be effector molecules in innate immunity, therefore serving as the body’s first line of defence (Boman, 1995Go). One group of peptide antibiotics is the cathelicidin family. The human cationic antimicrobial protein (hCAP-18) is the only known member of this family of proteins in man (Larrick et al., 1994Go; Cowland et al., 1995Go). The antimicrobial peptide LL-37 becomes activated when released from the C-terminal end of the hCAP-18 holoprotein. LL-37 has a broad antimicrobial activity against both Gram-positive and Gram-negative bacteria, and also against many fungal species (Bals et al., 1998Go). It is synthesized in the bone marrow in progenitors of the neutrophil series of differentiation and is stored as an 18 kDa protein in specific, peroxidase-negative granules of mature neutrophils (Sorensen et al., 1997aGo). The gene coding for hCAP-18, CAMP (cathelicidin antimicrobial peptide), is also expressed in the epithelium of other tissues such as the airway epithelium (Bals et al., 1998Go), mouth, tongue, oesophagus, vagina, cervix (Frohm Nilsson et al., 1999Go) and epididymis (Malm et al., 2000Go). In addition to its antimicrobial activity, LL-37 has chemotactic activity for neutrophils, monocytes and T cells (Agerberth et al., 2000Go; De et al., 2000Go). LL-37 interacts with the formyl peptide receptor-like 1 receptor on the surface of these cells (De et al., 2000Go). LL-37 may thereby contribute to both innate and adaptive immunity, the latter by recruiting immunocompetent cells to sites of microbial invasion.

We have recently shown that hCAP-18 is expressed in the male reproductive system (Malm et al., 2000Go). High levels were found in seminal plasma and hCAP-18 was also associated with sperm. High expression of the hCAP-18 gene was demonstrated in the epithelium of epididymis.

In this study, hCAP-18 was purified from seminal plasma and its identity confirmed by N-terminal amino acid sequencing. We also show that hCAP-18 appears in two major peaks after gel filtration of seminal plasma. hCAP-18 in the high molecular peak represents hCAP-18 bound to prostasomes, small membrane-bound vesicles derived from prostate epithelial cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Semen samples and preparation of prostasomes
Freshly ejaculated semen from healthy volunteers was collected at the Fertility Center, University Hospital MAS, Malmö, Sweden and the Department of Growth and Reproduction, Copenhagen University Hospital, Denmark. After semen liquefaction for 1 h at room temperature, the ejaculates were centrifuged for 20 min at 1000 g at room temperature in order to separate sperm from seminal plasma. The pellet was suspended in 0.1 ml, 30 mmol/l Tris–HCl buffer, pH 7.6, containing 0.13 mol/l NaCl. Cellular debris was pelleted by centrifugation at 10 000 g for 10 min at room temperature. The prostasomes were separated from seminal plasma by ultracentrifugation for 2 h at 109 000 g at room temperature. The pellet was resuspended in 0.1 ml 30 mmol/l Tris–HCl, 0.13 mol/l NaCl, pH 7.6. Sperm and prostasomes were washed three times in 1 ml of the same buffer.

Isolation of hCAP-18 from seminal plasma and N-terminal amino acid sequencing
An immunoglobulin fraction of rabbit anti-hCAP-18 antiserum (Sorensen et al., 1997bGo) was coupled to Affi 10 gel (BioRad, Hercules, CA, USA), 7 mg antibody/ml gel. A fresh sample of seminal plasma (4 ml) diluted 1:3 in equilibration buffer [50 mmol/l Tris–HCl, 0.5 mol/l NaCl, 0.1% Triton-X 100 (v/v), pH 7.5] was applied to the affinity chromatography column. The bound protein was eluted by lowering the pH [0.1 mol/l glycine, 0.5 mol/l NaCl, 0.1% Triton-X 100 (v/v), pH 2.2]. The fractions were analysed by Western blotting using rabbit anti-hCAP-18 antibody. Fractions containing hCAP-18 were pooled and concentrated using a Centricon 3 (Millipore, Bedford, MS, USA) centrifugal filter. The protein present in the pooled and concentrated fractions was cleaved by pyroglutamate aminopeptidase (EC 3.4.19.3; Sigma, St Louis, MO, USA) to remove pyroglutamate before Edman degradation. Thirty units of the enzyme were incubated with 2.7 µg hCAP-18 in 100 mmol/l Na2HPO4, pH 8.0, 10 mmol/l EDTA, 5 mmol/l dithiothreitol (ICN Biomedicals Inc., Aurora, OH, USA) and 5% (v/v) glycerol for 16 h at 50°C. After cleavage, the sample was run on sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) (see below) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The 18 kDa doublet band was cut out and N-terminal amino acid sequencing performed (Procise 494 Protein Sequencer; Applied Biosystems).

Analysis for detection of glycosylation of hCAP-18
The purified hCAP-18 was investigated by using the DIG Glycan/Protein Double Labeling Kit (Biochemica Boehringer Mannheim, Mannheim, Germany). In brief, the purified hCAP-18 was subjected to electrophoresis on a SDS–PAGE (Laemmli, 1970Go) and the protein was then transferred onto a nitrocellulose membrane (Towbin et al., 1979Go). Staining of the blotted protein was performed according to the manufacturer’s instructions.

Gel filtration of seminal plasma and immunoblots
Seminal plasma was diluted in 1 volume of phosphate-buffered saline (PBS). A 200 µl sample was applied to a Superose 12 column (Pharmacia, Uppsala, Sweden). The gel filtration was run under non-denaturing conditions. Fractions of 0.5 ml were collected and analysed with an hCAP-18 ELISA (Sorensen et al., 1997bGo). To investigate a possible co-localization of prostasomes and hCAP18, an antibody against the prostasome marker dipeptidyl peptidase IV (CD26) (Schrimpf et al., 1999Go) was used for immunoblotting (Dakopatts, Glostrup, Denmark). A total of 6 µl of each fraction was dotted onto PVDF membranes (Millipore). After blocking in skimmed milk [3% (w/v) in 10 mmol/l Tris–HCl, pH 8.0, 150 mmol/l NaCl, 0.05% Tween 20, (TBST)] the membranes were washed and CD26 visualized using monoclonal mouse anti-human CD26 antibody (2 µg/ml; Dakopatts) and porcine anti-mouse IgG conjugated with alkaline phosphatase (Promega Corp., Madison, WI, USA) as a secondary antibody. Bound antibodies were visualized by nitrobluetetrazolium/5-bromo-4-chloro-3-indolylphosphate (Sigma) in developing buffer (100 mmol/l Tris–HCl, 100 mmol/l NaCl, 5 mmol/l MgCl2, pH 9.5).

ELISA
A previously described (Sorensen et al., 1997bGo) sandwich ELISA was used to quantify hCAP-18 in the fractions after gel filtration.

Flow cytometry
Prostasomes were investigated for the presence of surface-associated hCAP-18 by flow cytometry (FACScan; Becton Dickinson, San José, CA, USA). The freshly prepared prostasomes were washed extensively with PBS (50 mmol/l, pH 7.5) containing 0.1% bovine serum albumin and thereafter incubated with the primary antibody. Rabbit anti-hCAP-18 antibody (0.2 µg) was used against an amount of prostasomes corresponding to ~90 µl human semen. After repeated washings, bound hCAP-18 antibodies were detected by a secondary fluorescein isothiocyanate-labelled anti-rabbit IgG antibody (Dakopatts). The prostasomes were neither fixed nor permeabilized before analysis in the flow cytometer. As a negative control, the primary antibody was replaced with an irrelevant isotype-matched factor V antibody (kindly provided by Professor Björn Dahlbäck, Malmö, Sweden).

SDS–PAGE and Western blot
SDS–PAGE (Laemmli, 1970Go) and immunoblotting (Towbin et al., 1979Go) were performed with BioRad systems according to the manufacturer’s instructions. Samples of 1.65x106 sperm, prostasomes corresponding to the amount in 16 µl semen and 5 µl of 1:10 diluted ultracentrifuged seminal plasma were analysed on a 15% SDS–polyacrylamide gel under reducing conditions. After protein transfer, the PVDF membranes (Millipore) were blocked for 30 min with 3% (w/v) skimmed milk in TBST. To detect hCAP-18, the membranes were incubated for 60 min with rabbit anti-human hCAP-18 antibody (2 µg/ml). After washing three times in TBST, incubation for 30 min with porcine anti-rabbit IgG conjugated with alkaline phosphatase (Promega) followed. The following washing steps and the visualization of bands on the membranes were performed in the same manner as described for the immunoblot experiment.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Isolation and N-terminal amino acid sequencing of hCAP-18 from seminal plasma
Affinity chromatography was performed using a polyclonal rabbit anti-hCAP-18 antibody. Fresh seminal plasma was applied to the column and, after washing, hCAP-18 was eluted. Fractions containing hCAP-18 were pooled and concentrated. As judged from SDS–PAGE, the eluted hCAP-18 was >95% pure. From a single semen sample ~25 µg hCAP-18 was purified, giving a total yield of ~7%. N-terminal amino acid sequencing of the doublet band was performed after removal of pyroglutamate. Thirteen steps were run, and 10 residues characterized corresponded to the earlier published sequence of hCAP-18 obtained from neutrophil granulocytes (Agerberth et al., 1995Go; Cowland et al., 1995Go; Larrick et al., 1995Go) (Figure 1Go).



View larger version (9K):
[in this window]
[in a new window]
 
Figure 1. Isolation of hCAP-18 from seminal plasma by affinity chromatography. The sequence obtained (A) was compared with the previously published sequence of hCAP-18 from neutrophil granulocytes (B). The affinity chromatography purified protein displayed sequence identity with hCAP-18. The arrow indicates removal of pyroglutamate (<Q) by pyroglutamate aminopeptidase, before performing N-terminal amino acid sequencing.

 
Dual distribution of hCAP-18 in seminal plasma
Fresh seminal plasma was subjected to gel filtration and the concentration of hCAP-18 in each fraction was measured (Figure 2Go). There was a large variation in the total concentration of hCAP-18 in seminal plasma between donors (mean 48 µg/ml, range 12–121; n = 6). After gel filtration, two peaks appeared, one of high and one of low molecular weight (Figure 2Go). Irrespective of the total amount of hCAP-18, approximately the same percentage of hCAP-18 was found in each molecular form in all samples. Approximately 70% (range 64–78; n = 6) of the total hCAP-18 in the sample was found in the high molecular peak and ~30% (range 22–36; n = 6) in the low molecular weight peak. The gel filtration fractions were also analysed for the prostasome marker dipeptidyl peptidase IV (CD26). Fractions in the first peak contained CD26 whereas fractions in the low molecular weight peak did not (circles below graph in Figure 2Go). This indicated that the high molecular weight peak contained prostasomes. The experiment was repeated with seminal plasma from five different individuals and essentially the same pattern was seen.



View larger version (11K):
[in this window]
[in a new window]
 
Figure 2. hCAP-18 in seminal plasma is found both as free monomeric protein and associated with prostasomes. A fresh sample of seminal plasma was applied on a Superose 12 column. Fractions were collected and the concentration of hCAP-18 measured by ELISA. All fractions were also analysed for immunoreactivity against the prostasome marker dipeptidyl peptidase IV (CD26) (circles below graph). Presence of dipeptidyl peptidase IV (CD26) was detected in fractions 14–22, corresponding to the high molecular peak of hCAP-18 and thus indicating that hCAP-18 is associated with prostasomes.

 
hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma
Pelleted sperm, prostasomes and ultracentrifuged seminal plasma were subjected to SDS–PAGE and Western blotting (Figure 3Go). Western blot analysis of a neutrophil homogenate, used as a positive control, showed a single band at 18 kDa corresponding to the hCAP-18 holoprotein. In contrast, a doublet band at 18 kDa was seen in all of the different semen components: sperm, prostasomes and ultracentrifuged seminal plasma (Figure 3Go). We were not able to detect any glycosylated forms of hCAP-18, as shown by using a staining protocol specific for glycoproteins (Biochemica Boehringer Mannheim). Therefore, the doublet band seen in Figure 3Go is unlikely to be due to glycosylation of the protein. Neither the cathelin fragment (14 kDa) nor the LL-37 peptide (4 kDa) was seen in the semen samples.



View larger version (116K):
[in this window]
[in a new window]
 
Figure 3. hCAP-18 immunoreactivity in sperm, prostasomes and ultracentrifuged seminal plasma as detected by SDS–PAGE and Western blotting. In a homogenate of neutrophil granulocytes (5x106 cells/ml, 10 µl sample corresponding to 50 000 cells were applied), a single band of ~18 kDa corresponding to hCAP-18 was detected (band 1). In sperm (1.65x106 sperm cells) (band 2), prostasomes, corresponding to the amount in 16 µl semen (band 3) and ultracentrifuged seminal plasma, diluted 1:10, 5µl (band 4) a corresponding doublet band is seen. The upper band corresponds to a molecular weight of 18 kDa whereas the lower band corresponds to a slightly smaller size.

 
Detection of hCAP-18 associated with the surface of prostasomes
Freshly prepared prostasomes from five different donors were analysed using a FACScan flow cytometer. The cytogram suggested that hCAP-18 was present on the surface of prostasomes. The positive signal shown when prostasomes were exposed to hCAP-18-specific primary antibody (Figure 4AGo) was not seen when the hCAP-18 antibody was replaced with an isotype-matched irrelevant antibody (Figure 4BGo).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 4. hCAP-18 is attached to the surface of prostasomes. By using flow cytometry, prostasomes were gated using their characteristics in forward and side scatter. (A) Bound hCAP-18 antibody, on the surface of prostasomes, was detected by a secondary fluorescein isothiocyanate-conjugated antibody indicating the presence of hCAP-18. (B) Replacement of the specific primary antibody with an irrelevant isotype-matched antibody at the same concentration resulted in a much lower signal, representing background labelling. The data obtained is from one out of five separate experiments with prostasomes from five different donors and essentially the same cytograms were seen.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study we have isolated hCAP-18 from seminal plasma and the identity of the protein was confirmed with N-terminal amino acid sequencing. Furthermore, we have shown that hCAP-18 has a dual distribution in seminal plasma. Gel filtration of seminal plasma, run under non-denaturing conditions, resulted in two distinct peaks of hCAP-18. The hCAP-18 in the high molecular peak can be explained by the association of hCAP-18 with prostasomes, while the low molecular weight peak is unbound hCAP-18. Comparison of free and prostasome-associated hCAP-18 with sperm-associated hCAP-18 (Malm et al., 2000Go) indicate that they are identical with respect to molecular size and immunoreactivity. Neutrophil homogenates were used as a positive control because of their previously described content of hCAP-18. Neutrophils contain 0.627 µg hCAP-18/106 cells (Sorensen et al., 1997bGo) compared with 0.196 µg of hCAP-18/106 sperm (Malm et al., 2000Go).

Prostasomes are complex membrane-bound corpuscular organelles produced by prostate epithelial cells. These organelles are expelled with the prostate secretions at ejaculation and can be purified from seminal plasma (Ronquist et al., 1978aGo,bGo). Prostasomes have a diameter of ~150 nm (Ronquist et al., 1990Go) and are claimed to have several functions. For example, prostasomes possess immunosuppressive capacity (Kelly et al., 1991Go; Skibinski et al., 1992Go) and after fusion with sperm (Arienti et al., 1997Go), they enhance sperm motility (Stegmayr and Ronquist, 1982Go). The biochemical background behind these effects is still unknown. Apart from hCAP-18, some other seminal plasma proteins have been found in association with prostasomes, e.g. tissue factor (Fernandez et al., 1997Go), the complement-regulatory protein membrane cofactor protein (CD46) (Simpson and Holmes, 1994Go; Kitamura et al., 1995Go) and membrane attack complex inhibitory protein CD59 (Rooney et al., 1993Go). The finding that hCAP-18 is associated with prostasome membranes is analogous to the finding that hCAP-18 is bound to the lipid part of lipoproteins in blood plasma (Sorensen et al., 1999Go). Essentially all hCAP-18 in blood plasma is bound to lipoproteins. Prostasome-associated hCAP-18 accounts for ~70% of all hCAP-18 in seminal plasma. The association of hCAP-18 with lipoproteins has been characterized as a non-covalent, hydrophobic interaction, with the cationic C-terminal end of LL-37 associated with the lipoprotein (Sorensen et al., 1999Go). It is therefore reasonable to believe that hCAP-18 in seminal plasma is associated with the prostasome membrane in the same way. This is also supported by the fact that LL-37 interacts with lipid bilayers (Turner et al., 1998Go).

The antimicrobial activity seen in prostasomes (Carlsson et al., 2000Go) could be due to hCAP-18, possibly in interaction with other antimicrobial peptides in seminal plasma such as the C terminal fragment of chromogranin B, secretolytin (Strub et al., 1995Go; Stridsberg et al., 1996Go). Expression of the CAMP gene and the large amount of hCAP-18 found in the epididymis (Malm et al., 2000Go) indicate its importance, probably in local defence against potentially invasive pathogenic micro-organisms. hCAP-18 has also been described intracellularly in the acrosome of human sperm (Hammami-Hamza et al., 2001Go), and this hCAP-18 is assumed to originate from the epithelial cells of testis (Hammami-Hamza et al., 2001Go). It is, however, possible that hCAP-18 attached to sperm (Malm et al., 2000Go) originates from prostasomes which have fused with sperm. The binding of hCAP-18 to prostasomes instead of directly to sperm could be a way of protecting sperm cells from the cytotoxic effects of LL-37. LL-37 could be cytotoxically active against prokaryotes and not eukaryotic cells due to the absence of cholesterol in the plasma membrane of prokaryotes. The plasma membrane of sperm possesses only a low cholesterol content (Mack et al., 1986Go) compared with prostasome membranes (Arvidson et al., 1989Go). When prostasomes, with associated hCAP-18, are fused with sperm, the sperm membranes will be enriched in cholesterol and the sperm could subsequently become resistant to the toxicity of LL-37.

Several studies have shown that human semen has antimicrobial activity. The antimicrobial capacity has been ascribed to different components of semen, for example lysozyme (Mardh and Colleen, 1974Go), spermine (Williams-Ashman and Lockwood, 1970Go) and the high zinc concentration (Mardh and Colleen, 1975Go). A possible reason for the relatively weak antimicrobial effect of seminal fluid is that hCAP-18 is present predominantly in its inactive proform with the C-terminal part, LL-37, still bound to the cathelin region. Activation may take place in the uterus. About 45 min after the sperm have entered the uterus, there is an extensive invasion of granulocytes from the female. These granulocytes release high concentrations of proteases, such as elastase and proteinase 3, which kill most of the sperm cells (Phillips and Mahler, 1977Go). Both elastase (data not shown) and proteinase 3 (Sorensen et al., 2001Go) have been shown to activate hCAP-18. The neutrophil granulocytes also contribute LL-37, since hCAP-18 is released from its specific granules in parallel with the release of proteinase 3 from the azurophilic granules (Sorensen et al., 2001Go). The physiological importance of semen-mediated and prostasome-mediated antimicrobial effects is easily envisaged. Both Gram-positive and Gram-negative bacteria and also some fungal species are found in the environment outside the cervix. We suggest that seminal plasma hCAP-18 is important for the protection of sperm cells in this unfriendly milieu. Free, uncomplexed hCAP-18 does not pass the cervix barrier and consequently does not enter the uterus. In contrast, hCAP-18 bound to sperm, probably transferred from prostasomes, may have its main function in the uterus or at conception in the oviduct. hCAP-18 may thus contribute to the sterile environment during fertilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are most grateful for the skilful technical assistance of Ingrid Wigheden and Hanne Kidmose. This study was supported by grants from the Alfred Österlund Foundation, the Greta & Johan Kock Foundations, the Th C Berg Foundation, the Swedish Asthma and Allergy Association’s Research Foundation, the Magnus Bergvall Foundation, the Malmö University Hospital Cancer Foundation, the Swedish Research Council, Scania County Council’s Research and Development Foundation, Foundation of University Hospital Malmö, Governmental Funding for Clinical Research and Fundacion Federico S.A.


    Notes
 
4 To whom correspondence should be addressed. E-mail: johan.malm{at}klkemi.mas.lu.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Agerberth, B., Gunne, H., Odeberg, J., Kogner, P., Boman, H.G. and Gudmundsson, G.H. (1995) FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc. Natl Acad. Sci. USA, 92, 195–199.[Abstract]

Agerberth, B., Charo, J., Werr, J., Olsson, B., Idali, F., Lindbom, L., Kiessling, R., Jornvall, H., Wigzell, H. and Gudmundsson, G.H. (2000) The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood, 96, 3086–3093.[Abstract/Free Full Text]

Arienti, G., Carlini, E. and Palmerini, C.A. (1997) Fusion of human sperm to prostasomes at acidic pH. J. Mem. Biol., 155, 89–94.[ISI][Medline]

Arvidson, G., Ronquist, G., Wikander, G. and Ojteg, A.C. (1989) Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering. Biochem. Biophys Acta, 984, 167–173.[ISI][Medline]

Bals, R., Wang, X., Zasloff, M. and Wilson, J.M. (1998) The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc. Natl Acad. Sci. USA, 95, 9541–9546.[Abstract/Free Full Text]

Boman, H.G. (1995) Peptide antibiotics and their role in innate immunity. Ann. Rev. Immunol., 13, 61–92[ISI][Medline]

Carlsson, L., Pahlson, C., Bergquist, M., Ronquist, G. and Stridsberg, M. (2000) Antibacterial activity of human prostasomes. Prostate, 44, 279–286.[ISI][Medline]

Cowland, J.B., Johnsen, A.H. and Borregaard, N. (1995) hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules. FEBS Lett., 368, 173–176.[ISI][Medline]

De, Y., Chen, Q., Schmidt, A.P., Anderson, G.M., Wang, J.M., Wooters, J., Oppenheim, J.J. and Chertov, O. (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes and T cells. J. Exp. Med., 192, 1069–1074.[Abstract/Free Full Text]

Fearon, D.T. and Locksley, R.M. (1996) The instructive role of innate immunity in the acquired immune response. Science, 272, 50–53.[Abstract]

Fernandez, J.A., Heeb, M.J., Radtke, K.P. and Griffin, J.H. (1997) Potent blood coagulant activity of human semen due to prostasome-bound tissue factor. Biol. Reprod., 56, 757–763.[Abstract]

Frohm Nilsson, M., Sandstedt, B., Sorensen, O., Weber, G., Borregaard, N. and Stahle-Backdahl, M. (1999) The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect. Immun., 67, 2561–2566.[Abstract/Free Full Text]

Hammami-Hamza, S., Doussau, M., Bernard, J., Rogier, E., Duquenne, C., Richard, Y., Lefevre, A. and Finaz, C. (2001) Cloning and sequencing of SOB3, a human gene coding for a sperm protein homologous to an antimicrobial protein and potentially involved in zona pellucida binding. Mol. Hum. Reprod., 7, 625–632.[Abstract/Free Full Text]

Kelly, R.W., Holland, P., Skibinski, G., Harrison, C., McMillan, L., Hargreave, T. and James, K. (1991) Extracellular organelles (prostasomes) are immunosuppressive components of human semen. Clin. Exp. Immunol., 86, 550–556.[ISI][Medline]

Kitamura, M., Namiki, M., Matsumiya, K., Tanaka, K., Matsumoto, M., Hara, T., Kiyohara, H., Okabe, M., Okuyama, A. and Seya, T. (1995) Membrane cofactor protein (CD46) in seminal plasma is a prostasome-bound form with complement regulatory activity and measles virus neutralizing activity. Immunology, 84, 626–632.[ISI][Medline]

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Larrick, J.W., Hirata, M., Zheng, H., Zhong, J., Bolin, D., Cavaillon, J.M., Warren, H.S. and Wright, S.C. (1994) A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity. J. Immunol., 152, 231–240.[Abstract/Free Full Text]

Larrick, J.W., Hirata, M., Balint, R.F., Lee, J., Zhong, J. and Wright, S.C. (1995) Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein. Infect. Immun., 63, 1291–1297.[Abstract]

Mack, S.R., Everingham, J. and Zaneveld, L.J. (1986) Isolation and partial characterization of the plasma membrane from human spermatozoa. J. Exp. Zool., 240, 127–136.[ISI][Medline]

Malm, J., Sorensen, O., Persson, T., Frohm-Nilsson, M., Johansson, B., Bjartell, A., Lilja, H., Stahle-Backdahl, M., Borregaard, N. and Egesten, A. (2000) The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa. Infect. Immun., 68, 4297–4302.[Abstract/Free Full Text]

Mardh, P.A. and Colleen, S. (1974) Lysozyme in seminal fluid of healthy males and patients with prostatitis and in tissues of the male uro-genital tract. Scand. J. Urol. Nephrol., 8, 179–183[ISI][Medline]

Mardh, P.A. and Colleen, S. (1975) Antimicrobial activity of human seminal fluid. Scand. J. Urol. Nephrol., 9, 17–23[ISI][Medline]

Phillips, D.M. and Mahler, S. (1977) Leukocyte emigration and migration in the vagina following mating in the rabbit. Anat. Rec., 189, 45–59.[ISI][Medline]

Ronquist, G., Brody, I., Gottfries, A. and Stegmayr, B. (1978a) An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid: part I. Andrologia, 10, 261–272.[ISI][Medline]

Ronquist, G., Brody, I., Gottfries, A. and Stegmayr, B. (1978b) An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid: part II. Andrologia, 10, 427–433.[ISI][Medline]

Ronquist, G., Nilsson, B.O. and Hjerten, S. (1990) Interaction between prostasomes and spermatozoa from human semen. Arch. Androl., 24, 147–157[ISI][Medline]

Rooney, I.A., Atkinson, J.P., Krul, E.S., Schonfeld, G., Polakoski, K., Saffitz, J.E. and Morgan, B.P. (1993) Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes and inhibits complement-mediated lysis. J. Exp. Med., 177, 1409–1420.[Abstract]

Schrimpf, S.P., Hellman, U., Carlsson, L., Larsson, A., Ronquist, G. and Nilsson, B.O. (1999) Identification of dipeptidyl peptidase IV as the antigen of a monoclonal anti-prostasome antibody. Prostate, 38, 35–39.[ISI][Medline]

Simpson, K.L. and Holmes, C.H. (1994) Presence of the complement-regulatory protein membrane cofactor protein (MCP, CD46) as a membrane-associated product in seminal plasma. J. Reprod. Fertil., 102, 419–424.[Abstract]

Skibinski, G., Kelly, R.W., Harkiss, D. and James, K. (1992) Immunosuppression by human seminal plasma—extracellular organelles (prostasomes) modulate activity of phagocytic cells. Am. J. Reprod. Immunol., 28, 97–103.[ISI][Medline]

Sorensen, O., Arnljots, K., Cowland, J.B., Bainton, D.F. and Borregaard, N. (1997a) The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood, 90, 2796–2803.[Abstract/Free Full Text]

Sorensen, O., Cowland, J.B., Askaa, J. and Borregaard, N. (1997b) An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma. J. Immunol. Methods, 206, 53–59.[ISI][Medline]

Sorensen, O., Bratt, T., Johnsen, A.H., Madsen, M.T. and Borregaard, N. (1999) The human antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma. J. Biol. Chem., 274, 22445–22451.[Abstract/Free Full Text]

Sorensen, O.E., Follin, P., Johnsen, A.H., Calafat, J., Tjabringa, G.S., Hiemstra, P.S. and Borregaard, N. (2001) Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood, 97, 3951–3959.[Abstract/Free Full Text]

Stegmayr, B. and Ronquist, G. (1982) Stimulation of sperm progressive motility by organelles in human seminal plasma. Scand. J. Urol. Nephrol., 16, 85–90[ISI][Medline]

Stridsberg, M., Fabiani, R., Lukinius, A. and Ronquist, G. (1996) Prostasomes are neuroendocrine-like vesicles in human semen. Prostate, 29, 287–295.[ISI][Medline]

Strub, J.M., Garcia-Sablone, P., Lonning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. and Metz-Boutigue, M.H. (1995) Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614–626 with antibacterial activity. Eur. J. Biochem., 229, 356–368.[Abstract]

Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 4350–4354.[Abstract]

Turner, J., Cho, Y., Dinh, N.N., Waring, A.J. and Lehrer, R.I. (1998) Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob. Agents Chemother., 42, 2206–2214.[Abstract/Free Full Text]

Williams-Ashman, H.G. and Lockwood, D.H. (1970) Role of polyamines in reproductive physiology and sex hormone action. Ann. NY Acad. Sci., 171, 882.[ISI]

Submitted on March 1, 2002; resubmitted on April 19, 2002; accepted on May 9, 2002.