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* Department of Immunology and Bacteriology, Western Infirmary, and
Center for Rheumatic Diseases, Royal Infirmary, Glasgow, United Kingdom;
Pharming Technologies, Leiden, The Netherlands; and
Instituto Finlay, Havana, Cuba
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
and less IL-5 and IL-10 upon stimulation ex vivo
with the exotoxin toxic shock syndrome toxin-1 compared with
congenic controls. To confirm that these effects of Lf transgene
expression could occur in the absence of live bacterial infection, we
also showed that Lf-transgenic DBA/1 mice exhibited enhanced severity
of collagen-induced arthritis, an established model of Th1-induced
articular inflammation. Higher levels of stainable iron in the spleens
of transgenic mice correlated with human Lf distribution, but all other
parameters of iron metabolism did not differ between transgenic mice
and wild-type littermates. These results demonstrate that human Lf can
mediate both antimicrobial and immunomodulatory activities with
downstream effects on the outcome of immune pathology in infectious and
inflammatory disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously investigated the role of Lf in Staphylococcus aureus infection, using a murine septic arthritis model (11). The appearance of multiple drug resistance has reawakened interest in natural host defense mechanisms against this organism. In particular, S. aureus is the most frequent etiological agent implicated in septic arthritis, which is characterized by severe articular destruction often complicated by septicemia with associated morbidity and mortality (12, 13). The disease can be reproduced in a murine model in which i.v. injection of an exotoxin-producing strain of S. aureus leads to development of severe septic arthritis, morbidity, and septicemia (14). Using this model, we have shown that local (periarticular) injection of human Lf (hLf) can reduce the severity of both joint inflammation (11) and collagen-induced arthritis (CIA), a model of human autoimmune arthritis. It was hypothesized that Lf acts locally by scavenging potentially damaging free iron from the inflamed joint. However, Lf could also exert a more general effect on immune responses in these models, but this was not investigated.
To address the possible role of hLf as an
immunomodulatory molecule, we have examined the response to S.
aureus of mice carrying a functional hLf gene. These
mice express Lf at high levels in milk, but also in other tissues at
lower levels (P. van Berkel and R. W. de Winter, unpublished
observations). Thus, hLf is present systemically and constitutively in
these mice, which means that it could influence the type of immune
response that occurs in the initial stages of infection. We demonstrate
here that the hLf-transgenic mice showed enhanced production of IFN-
and TNF- and reduced IL-5 and IL-10 production by spleen cells in
vitro, indicating enhanced Th1 polarization of the cell-mediated immune
response in the hLf-transgenic mice. This was associated with increased
ability to clear the bacteria and a trend to reduced development of
septicemia, arthritis, and mortality compared with their wild-type
littermates. In contrast, the development of CIA, in which Th1
responses exacerbate the early stages of disease (15), was
more severe in the transgenic animals. Together, these results indicate
that Lf promotes a Th1 response, and that its therapeutic potential in
infectious and inflammatory conditions depends upon the nature of the
immune polarization in disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hLf-transgenic mice (line 2261) and wild-type littermates (strain B6CBA) generated as previously described (16, 17) were used to establish a colony at Glasgow University (Glasgow, U.K.). For the CIA model, the transgenic mice were backcrossed on to the DBA/1 background and used after six backcrosses. In all cases expression of the transgene was monitored by tail-typing, and nontransgenic littermates were used as controls in the experiments described below.
Infection with S. aureus
Infection with S. aureus was conducted as described previously (11). Briefly, adult hLf-transgenic mice and age- and sex-matched nontransgenic littermates were injected i.v. with S. aureus strain LS-1 (107 CFU, except where otherwise indicated), a strain previously shown to produce arthritis as well as septicemia and morbidity (14). Arthritis and paw thickness were monitored daily for 2 wk using an arthritic score on a scale of 0–3 as defined previously (11), and evidence of sepsis (e.g., hunched appearance, raised fur, reduced spontaneous movement, lethargy) was noted (18). Mice (n = 3 per group) were killed before or 1, 2, 3, or 7 days after i.v. staphylococcal injection, and tissues were removed for histological, bacterial, biochemical, and immunological examination. The presence of bacteria in infected arthritic paws and the left kidney was determined as described previously (11). Preliminary experiments showed that these were the only tissues that contained an appreciable number of S. aureus.
Collagen-induced arthritis
Arthritis was induced as described previously (11). Briefly, hLf-transgenic mice on a DBA/1 background and age- and sex-matched nontransgenic littermates were injected intradermally with 0.2 mg bovine collagen in CFA, followed 3 wk later by another 0.2 mg i.p. injection (without CFA). Mice were monitored for arthritis as described above.
In vitro stimulation of spleen mononuclear cells for proliferation and cytokine production
Spleen single-cell suspensions, obtained by gently mincing spleens, followed by filtration through a cell strainer (BD Biosciences, Franklin Lakes, NJ), were cultured in U-bottom 96-well culture plates at 2 x 105 cells/well for up to 72 h at 37°C in RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (all from Life Technologies). Cells were stimulated with the staphylococcal exotoxins toxic shock syndrome toxin (TSST)-1 (10–100 ng/ml) and staphylococcal enterotoxin A (SEA; 100 ng/ml; both from Toxin Technology, Sarasota, FL) or heat-killed S. aureus (104 CFU/ml). Preliminary experiments were performed to establish the optimal concentrations of these stimulatory factors. Proliferation was determined by addition of 1 µCi [3H]thymidine (Amersham Life Sciences, Little Chalfont, U.K.) to triplicate wells 6 h before harvest, and cell 3H activity was counted in a beta counter (1205 Betaplate; Pharmacia Biotech, Uppsala, Sweden).
Determination of growth of S. aureus in vitro
A chemically defined medium (CDM) for growth of S. aureus (19) was rendered iron free by treating with Chelex-100 resin. A stock culture of S. aureus LS-1 was subjected to three passages on nutrient agar plates supplemented with 800 µM ethylenediaminedihydroxyacetic acid to reduce levels of stored iron, and a colony was then seeded into 1 ml CDM and cultivated overnight at 37°C. This culture was used to inoculate 25 ml CDM, with appropriate additions, at an initial OD of 0.1 at 470 nm, and growth was assessed spectroscopically. Iron was added as required in the form of iron-nitrilotriacetate, prepared by carefully mixing freshly prepared ferric chloride in 0.001 M HCl with a 4-fold molar excess of sodium nitrilotriacetate.
Immunoassays
Murine IFN-, IL-4, IL-5, and IL-10 were detected in
supernatants or plasma by ELISA with paired capture and biotinylated
detection mAbs (BD Biosciences, Oxford, U.K.) for each cytokine.
TNF- was detected with a mouse TNF- DuoSeT ELISA development
system (Genzyme, Cambridge, MA). The lower limit of detection in each
assay was 20 pg/ml. Human and mouse Lf were assayed by ELISA as
described previously (20), using in-house rabbit antisera
to human and mouse Lf. Preliminary experiments showed no immunological
cross-reactivity between human and mouse Lf.
Assessment of iron levels in tissues and plasma
Levels of hemoglobin and plasma and tissue non-heme iron were determined colorimetrically as described previously (21).
Immunohistochemistry
Perls Prussian blue reaction (for ferric iron). Paraffin sections were transferred to a fresh solution of equal parts of 2% aqueous potassium ferrocyanide and 2% hydrochloric acid for 30 min. Sections were washed several times in distilled water and counterstained lightly with 1% neutral red for 10 s. Finally, sections were washed in water, dehydrated, cleared, and mounted in DPX (Raymond A. Lamb, Eastbourne, U.K.).
Immunohistochemical staining. Frozen sections were defrosted and transferred to 10 mM sodium phosphate (pH 7.6). Sections were incubated for 30 min with blocking buffer (10 ml PBS, 2% pig serum, and 2% mouse serum). After this, rabbit anti-hLf was added for 60 min. Sections were washed, and the secondary biotinylated Ab (anti-rabbit IgG; Vector Laboratories, Burlingame, CA) was added for 30 min. Peroxidase activity was blocked by incubating the sections for 30 min in 0.3% H2O2 in methanol. Sections were incubated with Vectastain Elite ABC reagent (Vector Laboratories), washed in PBS, and incubated in peroxidase substrate solution until the desired stain intensity developed. Finally, sections were rinsed in tap water, counterstained with hematoxylin, cleared, and mounted in DPX.
Statistical analysis
Statistical analysis was performed using Student’s
t, Mann-Whitney, or 
2 test as
appropriate. Values of p < 0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasma cytokines.
Increased levels of IFN- were present in plasma 1 day after
infection, with lower levels detected from day 2, but there was no
significant difference between the groups (Fig. 1
). Significant elevation of plasma
TNF- was also observed in both hLf-transgenic and wild-type mice on
day 3 after infection, but again there was no significant difference
between the two groups (data not shown). No IL-4 or IL-5 expression was
detected in the plasma of any infected group.
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a), or heat-killed
S. aureus LS-1 (Fig. 2b). Significantly enhanced
proliferation to the SEA was also observed in transgenic mice after 2
days of infection (data not shown). In contrast, on day 0 the response
to TSST-1 was significantly greater in the wild-type mice, suggesting
that a priori the presence of hLf is immunomodulatory.
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(Fig. 3) than did spleen cells from wild-type
mice throughout the course of the experiment. IFN- production was
also significantly higher in cultures from hLf-transgenic mice than in
wild-type controls on days 3 and 7 after infection (Fig. 4). When cells were stimulated with SEA,
significantly enhanced IFN- production was also observed in
hLf-transgenic mice (data not shown). Because these data suggested type
1 T cell polarization, we next investigated the expression of cytokines
typical of type 2 responses. In contrast, IL-5 production was
significantly higher upon TSST-1 stimulation in cultures from wild-type
mice on day 1 after infection (Fig. 5a), and IL-10 production was
higher in cells from wild-type mice on day 3 (Fig. 5b; IL-4
levels were too low to be reliably assayed). These observations are
consistent with the presence of a dominant type 1 T cell response
during staphylococcal infection, which is further enhanced in
hLf-transgenic mice.
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a; the opposite
of the responses in infected mice), the immune responses in uninfected
mice were studied in more detail. Proliferation in response to TSST-1
was consistently greater in wild-type mice than in hLf transgenics at
all concentrations of TSST-1 (Fig. 6a). Moreover, IFN-
production in response to TSST-1 was consistently greater in spleen
cell supernatants of wild-type mice (Fig. 6b), and there was
also a significant difference, albeit less marked, in TNF-
production (Fig. 6c). Levels of IL-5 and IL-10 were too low
for reliable quantification. Thus, the enhanced type 1 T cell response
observed during infection in hLf-transgenic mice does not simply
reflect a priori type 1 polarization of responses to bacterially
derived superantigen.
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hLf-transgenic mice cleared the bacteria more readily than
wild-type mice (Table I); 28% of the
transgenics were free of bacteria in both kidney and joints (the only
tissues in which significant numbers of bacteria were found) at the end
of the experiment, compared with only 7% of wild-type mice
(p = 0.027, using 2
test). There was a trend to higher mortality evident in wild-type
(30%) compared with hLf-transgenic (21%) mice, although this was not
significant. Clinically detectable infectious arthritis at 10 days
postinoculation occurred in more wild-type than transgenic mice (27 vs
10%), although, as with mortality, this difference did not reach
significance (p = 0.12, using
2 test). The overall incidence of arthritis in
these C57BL background mice was much lower than that in Swiss mice
infected at the same time using the same protocol and bacterial load
(data not shown). Uninfected mice, both transgenics and wild type,
remained normal throughout.
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The above observations suggested that mice carrying the hLf transgene mounted a more pronounced Th1 response following S. aureus infection compared with controls, but that the effect on clinical outcome was less clear-cut, with only bacterial elimination achieving statistical significance. Resistance to S. aureus depends on both antimicrobial and anti-inflammatory activities; a Th1 response is beneficial for the former, but could exacerbate the latter, which might explain the lack of a clear protective effect of the hLf transgene.
To investigate this possibility, the effect of the hLf transgene on the
development of CIA was investigated. This Th1-driven arthritis lacks an
infectious component but reflects an autoimmune response to collagen.
It was found that the development of arthritis was indeed significantly
more severe in the transgenic mice (Fig. 7; p < 0.05).
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The above results clearly indicate that the systemic presence of
hLf has differing effects on S. aureus infection and CIA. A
likely explanation for the superior microbial clearance in the
transgenic mice during S. aureus infection is that hLf can
directly inhibit microbial growth. Therefore, the ability of hLf to
inhibit in vitro growth of S. aureus was tested. It was
found that hLf reduced the growth of S. aureus LS1 in a low
iron CDM, and this effect was reversible by iron (Fig. 8).
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There is some evidence that iron availability can affect the
polarization of T cell responses (22). Because Lf is an
iron-binding protein and can modulate iron supply to T cells
(6) as well as to bacteria, we investigated whether the
altered immunoregulatory function in hLf-transgenic mice was reflected
by changes in their iron metabolism. It has been previously reported
that serum levels of Lf are elevated in infection (23).
Transgenic mice expressed high levels of hLf in plasma (Table II), but no significant change in plasma
hLf levels was found on any day after infection. In contrast, plasma
levels of endogenous mouse Lf did indeed increase in both
hLf-transgenic and wild-type mice following infection. This presumably
reflects the fact that hLf is constitutively synthesized by various
tissues in the transgenic mice and is not influenced by neutrophil
degranulation, as would be the case for the endogenous mouse
Lf.
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, a and b) iron
deposits were observed in spleens from hLf-transgenic mice (Fig. 9a), but there was hardly any staining in wild-type spleens
(Fig. 9b). Iron accumulated mainly in the red pulp, possibly in
macrophages. This staining correlated with the distribution of hLf in
the spleens of the transgenic mice (Fig. 9c), while there
was no staining for hLf in the spleen of wild-type mice, as expected
(Fig. 9d). However, after 3 days of infection iron deposits
were seen in the spleen of both transgenic and wild-type mice (Fig. 9, e and f), and the distribution resembled that
seen in the uninfected transgenic mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that local administration of Lf into the
inflamed joints of mice with collagen-induced experimental arthritis
reduced inflammation (11). In these studies we did not
detect systemic modulation of Ag-specific immune responses. Because Lf
is an iron-biding protein that can interfere with iron uptake into T
cells (6), and because low iron availability is reported
to favor a Th1 response (22), Lf might be expected to
exert immunomodulatory effects in vivo. In this work we have
investigated the systemic effects of Lf on T cell responses to S.
aureus infection using transgenic mice carrying a functional
hLf gene. The casein promoter used in these mice results
primarily in expression in the mammary gland but is leaky in some mouse
lines (17, 25) (P. van Berkel and R. W. de Winter,
unpublished observations), permitting ectopic expression in other
tissues. This extramammary expression of Lf in the mouse line 2261 used
here was confirmed in this study, in which significant concentrations
of hLf (
400 ng/ml) were found in the plasma of these mice, and hLf
could be detected by immunocytochemistry in the spleen.
In S. aureus-infected mice it was found that
spleen cells from transgenic mice proliferated more strongly in
response to staphylococcal Ags either through conventional or
superantigen recognition pathways (TSST-1, SEA, or heat-killed
bacteria). The synthesis of IFN- and TNF- by these cells was also
increased in transgenic mice, whereas IL-5 and IL-10 synthesis was
higher in wild-type mice. This strongly suggests that hLf expression in
the transgenic mice enhances the Th1-type response that is normally
associated with S. aureus infection
(26). Because the Th1 response against S.
aureus is protective (27), it seems likely that this
enhancement is responsible for the improved bacterial clearance in the
transgenic mice. However, there is a two-edged nature to immune
response polarization in bacterial infection. T cell subsets bearing
V
11 TCR have been clearly implicated in septic arthritis disease
progression (28). Thus, protective Th1 responses may clear
bacteria but simultaneously exacerbate inflammatory responses.
Similarly, macrophages may be both antimicrobial and proinflammatory in
the mouse S. aureus arthritis model (29).
Commensurate with these observations, IFN- blockade reduces the
severity of septic arthritis in Swiss mice, directly implicating host
Th1 responses in clinically detectable murine arthritis, whereas
treatment with IFN- decreases mortality but enhances the development
of arthritis (30). This may explain why the incidence of
arthritis did not differ significantly between transgenic and congenic
controls in our studies. To further investigate this possibility, we
backcrossed the Lf transgene to the DBA/1 strain that develops chronic
arthritis associated with a Th1-polarized response to type II collagen
(CIA). We found that CIA in Lf-transgenic DBA/1 mice was characterized
by significantly increased severity of joint inflammation.
The ability of hLf to cause Th1 polarization did not reflect prior Th1-type predisposition of the transgenic mice. Whereas cells from infected transgenic mice showed an enhanced Th1 response to S. aureus Ags in vitro, the opposite effect was seen with cells from uninfected mice. Thus, TSST-1 and (at high concentrations) heat-killed S. aureus generated greater proliferation and type 1 cytokine production by wild-type thanby hLf-transgenic spleen cells. TSST-1 is known to act as a superantigen (31), and it may be that hLf has a dual action. In uninfected mice it has a general down-regulatory effect on T cell proliferation, as has been previously reported for humanT cells (6, 7), but once an infection with an associated Th1 response is established, hLf favors a Th1 over a Th2 response. The precise mechanism underlying this phenomenon remains unclear, but it may reflect interference with the increased iron requirement of T cells following activation (32).
We have previously reported that Lf administered
periarticularly to mice with joint inflammation, in both CIA or septic
arthritis, reduced the degree of joint inflammation (11).
This is in contrast to and apparently contradicts the increased joint
inflammation evident in hLf-transgenic mice with CIA. The mode of
delivery of Lf appears crucial to its function. Local administration of
hLf to animals with established joint inflammation is unlikely at that
stage to influence the systemic immune response. We have proposed that
the mode of action of locally administered Lf is via sequestration of
potentially toxic free iron in the inflamed joint, a mechanism that
would operate equally well in both septic arthritis and CIA
(20). In addition, Lf probably has a direct antibacterial
effect on S. aureus. It has long been known that Lf inhibits
the growth of many bacteria, including S. aureus, by virtue
of its ability to sequester iron and render it unavailable to
microorganisms (1), and indeed such an effect was found in
vitro with the LS-1 strain of S. aureus used in this work
(Fig. 8). The hLf-transgenic mice showed an enhanced ability to clear
S. aureus compared with wild-type littermates, and others
have shown that iv administration of hLf protected mice against
experimental kidney infection (33).
Sequestration of iron would be expected to exert quite distinct effects on systemic immune responses to the foregoing local activities. To assess whether the altered T cell responses in the hLf-transgenic mice were related to alterations in iron metabolism, we examined the iron status of these mice. No differences were found between transgenics and control mice with regard to plasma and liver iron or hemoglobin levels. The same was true with mice infected with S. aureus. This is perhaps not surprising, as Lf plays no role in the main pathways of mammalian iron metabolism, and it has recently been reported that Lf-knockout mice also display a normal iron phenotype (34). However, although total spleen iron measured chemically was similar in transgenic and wild-type mice, Perls-positive iron deposits were detected in the spleens of transgenic mice, but not in wild-type controls, and these colocalized with staining for hLf. This was observed in both infected and uninfected mice, and serum levels of hLf did not change upon infection of the transgenic animals. However, in wild-type mice in which splenic iron deposits were initially absent, Perls-positive staining appeared following infection, correlating with an increase in plasma levels of the endogenous mouse Lf. It is possible that accumulation of Lf in the spleen alters the relative distribution of iron within this organ, perhaps by removing it from the extracellular environment and depositing it in spleen cells such as macrophages, where it forms Perls-positive deposits. This could affect the initial immune response of spleen cells. However, the antimicrobial and immunomodulatory activities of Lf have also been ascribed to an iron-independent mechanism mediated through the basic N-terminal region of the Lf molecule (3), and the participation of such a mechanism in the present report cannot be ruled out.
Why should hLf in mice affect T cell polarization and the response to S. aureus when endogenous murine Lf already present might be expected to carry out similar functions? Murine Lf differs significantly from the human protein. In particular, it lacks the sequences of basic N-terminal amino acids that give rise to the microbicidal and anti-inflammatory effects of this region in human and bovine Lf (35). Moreover, its affinity for iron is lower than that of the human and bovine proteins (36), suggesting that it may function less well as an iron-binding protein in inflammatory foci. The biological role of Lf in the mouse may therefore differ from that in man. However, that fact that mouse Lf levels increased following infection, whereas hLf levels remained constant means that mouse Lf may also affect the response to S. aureus.
In conclusion, we have demonstrated that mice expressing hLf show modulation of their immune responses, manifest by polarization toward a Th1 response during active infection or Ag challenge. Lf has been proposed as a therapeutic agent. Our results clearly have important implications for such potential therapeutic use of Lf as an anti-inflammatory, antimicrobial, or immunomodulatory agent and indicate the necessity for a cautious approach in the first instance.
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Footnotes |
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2 Current address: Department of Immunology and Cell Biology, Mario Negri Institute of Pharmacological Research, Milan, Italy.
3 Address correspondence and reprint requests to Dr. Iain B. McInnes, University Department of Medicine, Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, U.K. E-mail address: i.b.mcinnes{at}clinmed.gla.ac.uk
4 Abbreviations used in this paper: Lf, lactoferrin; CDM, chemically defined medium; CIA, collagen-induced arthritis; TSST, toxic shock syndrome toxin; hLf, human Lf; SEA, staphylococcal enterotoxin A.
Received for publication July 5, 2001. Accepted for publication February 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 in transgenic mice increases mortality during infection by enterotoxin A-producing Staphylococcus aureus. Infect. Immun. 63:4463.[Abstract]
11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes. Immunology 94:1.[Medline]
in experimental Staphylococcus aureus septicaemia versus arthritis. Immunology 93:80.[Medline]
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) mice. All data are the mean ± SD of triplicate
culture pooled from three mice and are representative of two
experiments in which similar data were obtained.
) or supplemented with native (i.e., largely
iron-free) hLf (100 µg/ml; 
). The experiment was performed three
times; data are single readings from a representative experiment.