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*
Center for Microbial Pathogenesis, University of Connecticut Health Center, Farmington, CT 06030;
Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Portland, OR 97201;
Department of Pediatrics, Division of Pediatric Infectious Diseases, University of Connecticut School of Medicine, Connecticut Children’s Medical Center, Hartford, CT 06106;
Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and
¶ Department of Medicine, University of Connecticut School of Medicine, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Host defenses against microbial pathogens involve a complex interplay of innate and adaptive immunity (9). Innate immunity involves preprogrammed responses to diverse microbial constituents, whereas the slower adaptive immune response results in the generation of specific Abs and Ag-sensitized T cells. Although T. pallidum lacks LPS, the proinflammatory molecule found in the outer membranes of Gram-negative bacteria, it does contain abundant lipoproteins (10, 11, 12, 13). Evidence from both in vitro and in vivo studies have demonstrated that these lipid-modified proteins are potent activators of effector cells associated with innate immunity, principally monocytes/macrophages and endothelial cells (14, 15, 16, 17, 18, 19, 20). Because T. pallidum cannot be cultivated in vitro, investigation of the proinflammatory properties of treponemal lipoproteins has been hampered by difficulties in isolating sufficient quantities of these molecules. This obstacle has been circumvented by the use of synthetic lipohexapeptides that correspond to the N termini of the full-length proteins. A number of studies have shown that these lipoprotein surrogates possess proinflammatory properties qualitatively similar to those of their native counterparts (17, 18, 19, 20, 21). Most recently, we and others have demonstrated that cellular activation by native lipoproteins and synthetic analogues proceeds via the Toll-like receptor 2 (TLR2)3-dependent signaling pathway as opposed to LPS-mediated signaling, which uses TLR4 (22, 23, 24, 25, 26, 27).
The rabbit has been the traditional animal of choice for studying the evolution of histopathological changes and the development of cellular and humoral immune responses to T. pallidum during infection (2, 3). In a prior study (28), we extended this model to demonstrate that intradermal injection of treponemal lipopeptides elicits cellular infiltrates resembling those observed in acquired syphilis in humans. However, a detailed analysis of this response was precluded by the paucity of reagents directed against rabbit immune cells and immunomodulators. For this reason, we turned to an in vivo human skin model to characterize further the biological properties of T. pallidum lipoproteins and to define their role in disease pathogenesis. In this model (29), mild suction is used to elicit blister formation at the dermo-epidermal junction following intradermal injection with treponemal lipopeptides. In contrast with the paucity of cells in fluid from blisters raised over saline-injected skin or unmanipulated normal skin, fluid within blisters elicited over sites of inflammation contains numerous extravasated immune cells that can be analyzed by flow cytometry (30, 31, 32, 33); comparison with peripheral blood (PB) enables one to identify selectively recruited leukocyte subsets and to assess the influence of the inflammatory microenvironment on their state of activation/differentiation (31, 32, 33). There is now extensive evidence that the composition of cells in blister fluid accurately reflects the cellular infiltrates within the underlying dermis (30, 31, 32, 33).
We postulated that studying these agonists in skin, a major target organ of syphilitic infection (1), would yield insights into their biological activities that could not be obtained from in vitro investigations using highly purified leukocyte subtypes or leukocytic cell lines. Our findings demonstrate that treponemal lipopeptides have the capacity to induce in human skin an inflammatory milieu reminiscent of that found in early syphilis lesions. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 (34, 35) was a marked enhancement of CCR5 positivity among mononuclear cell populations, particularly macrophages, in the blister fluids. In contrast with in vitro studies that have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eligible participants were healthy volunteers between the ages of 18 and 60 years without clinical or serological evidence of syphilis. Individuals were considered ineligible if they were taking anti-inflammatory medications or had a history of chronic dermatoses. All participants underwent a physical examination before enrollment. Protocols used in this study were approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center and the University of Connecticut Health Center. Informed written consent was obtained from all persons who participated. A total of 45 subjects were enrolled for the various studies described in Results. The participants ranged from 20 to 54 years of age and included 22 males and 23 females, consisting of 41 Caucasians (including 3 Hispanics), 2 Blacks, and 2 Asians.
Synthetic treponemal lipohexapeptides and hexapeptides
Lipohexapeptides (lipopeptides) corresponding to the N termini
of the T. pallidum 17- and 47-kDa lipoproteins (designated
17-L and 47-L, respectively) were synthesized and extensively
characterized as described previously (21). The
corresponding nonlipidated hexapeptides (17 and 47,respectively)
also were synthesized as controls using standard
9-fluorenylmethoxycarbonyl chemistry on an Applied Biosystems
(Foster City, CA) 430A peptide synthesizer. Both hexapeptides and
lipopeptides contained undetectable levels of endotoxin (
1 pg
LPS/µg protein) as measured by the QCL-1000 quantitative, chromogenic
Limulus amebocyte lysate assay (BioWhittaker, Walkersville,
MD). For intradermal injection, lyophilized hexapeptides and
lipopeptides were suspended by vortexing in sterile
H2O for drug diluent use (Abbott Laboratories,
North Chicago, IL).
Intradermal injection of synthetic hexapeptides/lipopeptides and elicitation of epidermal blisters
To establish the dosage of lipopeptides for flow cytometric
studies, five volunteers were injected intradermally at four separate
sites on the volar surface of the forearm with 25, 50, and 100 µg of
17-L or 47-L and 100 µg of the corresponding hexapeptide.
Subsequently, volunteers were injected intradermally at separate sites
with 100 µg of each lipopeptide. Twenty-four hours later, the sites
were swabbed with alcohol and an acrylic suction cup was applied, as
shown in Fig. 1
A. A thin coating of high vacuum silicone
lubricant (Dow Corning, Midland, MI) on the underside of the suction
blister cup ensured an airtight seal with the surface of the skin.
Vacuum suction (200 mm Hg) and gentle warming with a 125 W infrared
lamp were used for 1.5–2 h to raise epidermal blisters (Fig. 1A, inset). Fluid was syringe aspirated from the
blisters the following day (48 h postinjection).
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Monoclonal and isotype-matched control Abs conjugated to FITC,
PE, PerCP, and allophycocyanin were obtained from Becton Dickinson
Immunocytometry Systems (BDIS, San Jose, CA), with the exception of
FITC-
-TCR (Endogen, Woburn, MA).
Cell staining and flow cytometry
Whole blood was either treated with erythrocyte lysis buffer
(150 mM NH4Cl, 1 mM KHCO3,
and 1 mM EDTA) to produce erythrocyte-depleted leukocytes or
centrifuged in Vacutainer Cell Preparation Tubes (Becton Dickinson,
Franklin Lakes, NJ) to isolate PBMCs. Blister fluids were transferred
to 3 ml of fluorescence assay (FA) buffer (Difco Laboratories, Detroit,
MI) containing 2 mM EDTA (pH 8) and washed once before resuspension in
0.5 ml of the same buffer. Following enumeration using a hemacytometer,
aliquots of 
5 x 104 freshly isolated
erythrocyte-depleted leukocytes, PBMCs, or blister fluid cells were
placed into 12 x 75 polypropylene tubes (Becton Dickinson
Labware, Lincoln Park, NJ) and washed once with FA buffer containing
0.1% BSA and 13 mM NaN3
(FA/BSA/NaN3), followed by resuspension in 50
µl of the same buffer. Cells then were blocked with 10 µg of
purified human IgG (Sigma, St. Louis, MO) for 15 min on ice, followed
by incubation with fluorochrome-conjugated mAbs for another 30 min
protected from light. Additionally, aliquots of erythrocyte-depleted
leukocytes were incubated with individual fluorochrome-conjugated Abs
or isotype-matched control Abs to compensate for fluorescence emission
overlap and nonspecific fluorescence, respectively. The intrinsic and
nonspecific fluorescence of negative cell populations was adjusted to
fall within the first decade, thus delineating positive cell
populations as those whose mean fluorescence intensity falls within the
second to fourth decade. Staining of blister fluid cells with
isotype-matched control Abs did not produce any greater nonspecific
fluorescence than that observed with erythrocyte-depleted leukocytes.
After staining, cells were washed once with
FA/BSA/NaN3 and fixed by resuspension in 1 ml of
FA buffer containing 1% paraformaldehyde (Electron Microscopy
Sciences, Fort Washington, PA) and 13 mM NaN3.
Fluorescence data were acquired on a FACSCalibur dual laser flow
cytometer (BDIS) using a threshold of 52 and an appropriate scatter
gate to exclude dead cells, cellular debris, and residual erythrocytes.
List mode multiparameter files (consisting of forward scatter,
orthogonal scatter, and three or four fluorescence parameters) were
analyzed using PAINT-A-GATEPRO (version 2.0)
software (BDIS). Using this program, events were sequentially
"painted" to identify up to seven discrete cell populations based
upon phenotypic signatures and their relationship to each other. These
populations then were quantified as percentages of the total events (or
a gated subset thereof), and their mean channel fluorescence
intensities were calculated.
Statistics
An ANOVA followed by a multiple comparisons test was used to determine whether significant differences existed between data sets. Significance was accepted when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the outset, we characterized the gross inflammatory response to
the lipopeptides and determined the dosages for subsequent flow
cytometric studies. Five volunteers were injected on the volar surface
of the forearm with graded doses (25, 50, and 100 µg) of 17-L or 47-L
and with 100 µg of the corresponding hexapeptide. In all five
subjects, dose-dependent erythema and induration were observed within
24 h following intradermal injection with either lipopeptide; this
response was maximal at 
48 h (Fig. 1
B) and resolved by 96 h
(data not shown). None of the subjects experienced significant
discomfort at any of the injection sites. In accordance with
prior studies showing that lipid modification isessential for
the proinflammatory activity of both full-lengthlipoproteins and
synthetic analogues (16, 17, 18, 21, 28), no responses were
observed at sites injected with hexapeptides (Fig. 1B).
Fluids obtained immediately after the blisters were raised were
essentially devoid of cells. However, 24 h later the time point
corresponding to the peak gross inflammatory response, dose-dependent
increases in white blood cells, and only rare erythrocytes were
observed at sites receiving lipopeptides (Table I). Consistent with the lack of gross
inflammatory response, sites injected with 100 µg of either
hexapeptide contained 100-fold fewer cells than those receiving an
equivalent amount of either lipopeptide (Table I). These results show
that the vast majority of cells in the blister fluids were elicited by
the lipopeptides. Based upon these findings, in all subsequent
experiments volunteers received 100 µg doses of 17-L and 47-L.
Blisters were raised over sites 24 h after injection, and the
fluids were aspirated the following day. Parallel injections with
hexapeptides were discontinued because the low numbers of cells
elicited precluded reproducible flow cytometric analysis.
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Flow cytometric studies were performed to identify the major
leukocyte populations in the blister fluids. Representative results for
a single individual are shown in Fig. 2, and a summary of the results is presented in Table II. The absence of significant
differences between the lipopeptides is in agreement with prior in
vitro studies showing comparable potencies and biological activities
for these agonists (17, 18, 19, 20, 21). Infiltrates elicited by the
lipopeptides consisted predominantly of neutrophils and
monocytes/macrophages, but also contained substantial numbers of
lymphocytes. Compared with PB, blister fluids were markedly enriched
(8-fold) for monocytes/macrophages, relatively deficient in T
lymphocytes, and virtually devoid of B lymphocytes, while the
percentages of granulocytes in the two compartments were similar.
Monocytic cells in blister fluids were larger and more granular by
forward and side scatter characteristics than their circulating
counterparts and expressed 3-fold more CD14 (Fig. 2) and HLA-DR
(data not shown) on their surfaces, findings that indicated that they
had differentiated into activated macrophages within the cutaneous
microenvironment. That monocytic cells increased their surface
expression of CD14 within inflamed skin also was noteworthy because it
contrasted with prior in vitro studies in which incubation of PB
monocytes with treponemal lipoproteins/lipopeptides had the opposite
effect on CD14 expression (20).
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). Only a minuscule percentage of T cells (<1%) in the
blister fluids were of the virgin (i.e.,
CD45RO-/CD95-) phenotype.
In contrast, roughly 70% of the T cells were
CD45ROhigh memory/effector cells, while the
remainder displayed the
CD45ROlow/CD95+
memory/effector phenotype that is associated with repeated antigenic
stimulation (36). Approximately 80% of the blister fluid
T cells stained positively for the skin-homing receptor cutaneous
lymphocyte Ag (CLA) (37), a 4-fold increase above the
percentage of CLA+ cells found in PB
(p < 0.001). The proportions of blister fluid
T lymphocytes that were positive for CD4, CD8, or the -TCR did
not differ significantly from those in PB, indicating that a bias for
recruitment of these subsets did not exist. Approximately 10-fold more
of the CLA+ blister fluid T cells expressed
HLA-DR than did their CLA+ counterparts in PB,
raising the possibility that a subpopulation of T cells was activated
within the proinflammatory environment created by the lipopeptides. CC
chemokines, particularly RANTES, which are produced in abundance by
lipoprotein/lipopeptide-activated macrophages (20), could
have been responsible for this effect (38). Lipopeptides promote recruitment of dendritic cells (DCs) and maturation of the myeloid- but not lymphoid-derived DC subset
DCs, professional APCs whose primary function is to capture and
process Ags for presentation to T and B cells (39, 40),
can be recruited to peripheral sites by a broad range of inflammatory
stimuli (41). Because of their unique function at the
interface between the innate and adaptive immune responses, we next
asked whether lipopeptides could recruit these potent inducer cells
into skin. DCs were identified in PB and blister fluids by their lack
of staining with FITC-conjugated lineage markers (CD3, CD14,
CD16, CD19, CD20, and CD56) and their positive staining for
HLA-DR. Although numerically minor components in both compartments, DCs
were between 5- and 10-fold more abundant in blister fluids than in PB,
differences that were highly significant (p <
0.001) (Fig. 3 and Table III). Staining for the
maturation/differentiation markers CD83 (42) and CD1a
(43) was next performed to assess the developmental state
of DCs in the two compartments. Neither Ag was detected on circulating
DCs, whereas both surface molecules were expressed by a
HLA-DRhigh subpopulation of cells in the blister
fluids (Fig. 3 and Table III).
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2:1) of DC1 and
DC2 subsets (Fig. 4A and Table III), a ratio comparable with that reported by others (43, 44, 47). Little is known about the trafficking patterns of DC2
cells, although there is evidence that they migrate from the
circulation into lymphoid tissues via high endothelial venules
(45, 49, 50). To our knowledge, this is the first report
in which DC2 cells were identified in extralymphoidal inflammatory
sites. With regard to DC1 cells, several differences were noted between
this subpopulation in PB and blister fluids. Compared with their
circulating counterparts, DC1 cells in the blister fluids 1) were
larger and more granular (data not shown), 2) expressed 2-fold
greater levels of CD11c, and 3) expressed 7-fold greater levels of
HLA-DR (Fig. 4A). The observation that expression of CD83
and CD1a was confined largely to a HLA-DRhigh
subpopulation suggested that these Ags were associated with the DC1
subset in the blister fluids (Fig. 3). This was confirmed directly by
staining blister fluid DCs for CD11c, CD83, and CD1a (Fig. 4B and Table III).
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, while representative flow cytograms are presented in Fig. 5. In accord with previous studies
(52), CD80 was not detected on DCs in PB. However, it was
strongly induced on most DC1, but not on DC2 cells, in the blister
fluids. The inducibility of CD80 expression on DCs also has been noted
previously (52), although, to our knowledge, not
specifically related to DC1 and DC2 subpopulations. The expression
patterns for CD86 were more complex. Consistent with prior reports
stating that CD86 is constitutively expressed by DCs in PB
(51), most circulating DCs expressed this molecule; it was
noteworthy that the highest levels of CD86 were expressed by the DC2
cells. Most of the DC1 cells from the cutaneous compartment expressed
CD86 at levels moderately or markedly greater than their PB
counterparts. The CD86high subset of DC2 cells
seen in PB was not evident in the blister fluids. In fact, all of the
blister fluid DC2 cells expressed relatively low levels of this
Ag.
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In a recent in vitro study (20), we demonstrated that
incubation of PBMCs with T. pallidum, treponemal
lipoproteins, or synthetic lipopeptides resulted in enhanced expression
of CCR5 and a reciprocal decrease in the expression of CXCR4. Based
upon these results, we proposed that up-regulation of CCR5, the
coreceptor for M-tropic strains of HIV-1 (35), in response
to T. pallidum and its lipoprotein constituents was a
potential biological correlate to the epidemiological observation that
M-tropic strains of the virus are typically involved in establishing
primary infection by the sexual route (53). To garner
additional support for this idea, as well as to shed light on the
role(s) these chemokine receptors might play in leukocyte trafficking
into inflamed skin, we compared the expression of CCR5 and CXCR4 by
mononuclear cells within the PB and cutaneous compartments. An increase
in the percentage of cells expressing CCR5 was observed for all three
mononuclear cell populations in blister fluid (Fig. 6A). This effect was most
pronounced for monocytic cells, in which marked increases in both the
percentages of cells expressing CCR5 and in their mean fluorescence
intensity occurred. Although the percentage of T cells expressing CCR5
was significantly increased in blister fluid, the intensity of CCR5
expression was unchanged, suggesting that lipopeptides promoted
selective recruitment of CCR5+ T cells into the
dermis. We confirmed this by finding that CLA+ T
cells in both compartments expressed identical levels of CCR5 (data not
shown). A modest, although statistically significant, increase in the
percentage of CCR5+ DCs in blister fluid also was
observed. However, more striking was the 3- to 4-fold increase in the
levels of this chemokine receptor expressed by blister fluid DCs. In
contrast to CCR5, the percentage and mean fluorescence intensity of
mononuclear cells within blister fluids expressing CXCR4 were either
decreased (monocytes/macrophages) or unchanged (T cells and DCs) (Fig. 6B). Finally, we also determined whether DC1 and DC2 cells
differed with respect to the expression of CCR5. Surprisingly, the
increase in the proportion of CCR5+ cells among
blister fluid DCs was confined to the DC2 subset (Fig. 7A), whereas both subsets
manifested significantly increased levels of CCR5 expression (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of innate immunity by microbial constituents, now frequently termed pathogen-associated molecular patterns or PAMPs (56), is considered a prerequisite for the development of the slower adaptive responses that are often essential for pathogen elimination (9, 56, 57). Indeed, in the rabbit model of experimental syphilis, clearance of treponemes correlates with the local influx of T cells and the appearance of opsonic Ab (2, 3). Therefore, a second major objective of our study was to examine the capacity of lipoprotein-mediated responses to bridge innate and adaptive immunity in cutaneous lesions of early syphilis. Along these lines, we found that two synthetic lipoprotein analogues were highly effective at recruiting cellular elements, DCs and memory/effector T lymphocytes, required for primary and/or secondary immune responses. Trafficking of DCs, as well as T cells, into skin is mediated by CLA and other isoforms of P-selectin glycoprotein ligand 1, which are expressed constitutively in PB (37, 58). Therefore, it is highly likely that the enrichment for CLA+ T cells and DCs in the blister fluids reflected the expression of receptors for these molecules (i.e., E- and P-selectin) by activated vascular endothelium. It also is plausible that the innate response to treponemal lipoproteins helps to set the stage for the Th1 bias observed in primary and secondary syphilis lesions (8, 59). DC1 cells, the subpopulation that expressed maturation markers and both costimulatory molecules within blister fluid, promote Th1 differentiation (48, 60) and would be expected to do so during infection when presenting processed treponemal Ags to naive T cells within draining lymph nodes. In support of this contention, spirochetal lipopeptides and the 19-kDa lipoprotein from Mycobacterium tuberculosis stimulate monocyte-derived DCs to release IL-12, which directs the selective development of a Th1 response (61). Furthermore, the expression of CCR5 on the surface of activated DCs has been shown to provide a signal for this microbial-induced production of IL-12 (62). Finally, both CLA+ and CCR5+ T lymphocytes, which were highly enriched in the blister fluids, also are associated with Th1-type reactions (63, 64, 65, 66, 67, 68).
Although increased numbers of DCs have been reported in different
tissue models of inflammation (41, 58, 69), the relative
importance of recruitment from blood vs resident tissue sources has not
been well established. For this reason, the source of DCs in the
blister fluids is an important question. We believe that the blister
fluid DCs were derived from both the PB and skin compartments. The fact
that PB and blister fluids consistently contained highly similar ratios
of DC1 and DC2 cells suggests that the overwhelming majority of DCs
extravasated from PB. At the same time, a small percentage of blister
fluid DCs expressed very high levels of CD1a (designated by arrows in
Fig. 3) and are likely to represent locally recruited, resident
Langerhans cells (43, 70). Inasmuch as monocytes can
differentiate into DC1 cells upon extended incubation with
monocyte-conditioned medium (71) or various combinations
of cytokines (e.g., IL-4, GM-CSF, TNF-) (49, 72, 73, 74, 75, 76),
one could argue alternatively that the DC1 subset actually arose from
infiltrating monocytes that differentiated in situ. Two lines of
evidence indicate that this is unlikely to be the case. One is that the
inflammatory milieu within the sites of injection induced monocytes to
mature into activated macrophages rather than DCs, as evidenced by
their increased expression of CD14, a surface marker lost when monocyte
progenitors become DCs (72, 73, 74, 75, 76). Second, in accord with
our in vivo findings, Randolph and coworkers (77) showed
using an elegant in vitro model that monocytes differentiate into
macrophages when they traverse endothelial monolayers, while only those
cells that subsequently egress in the basal-to-apical direction
differentiate into DCs.
In vitro studies of DC maturation and function typically use either
blood or bone marrow-derived progenitors that respond as a homogeneous
population to inflammatory stimuli, including LPS and other PAMPs
(78, 79, 80). However, in recent years it has become apparent
that circulating DCs are comprised of two distinct lineages denoted by
reciprocal expression of CD123 and CD11c, and that this heterogeneity
has profound implications for the induction of T cell differentiation
during the primary immune response (43, 44, 45, 46, 48, 49, 60, 81). Therefore, a striking observation was the remarkable
dichotomy in differentiation potential displayed by these two DC
lineages within the inflammatory milieu induced by the lipopeptides. At
first blush, one might conclude from the absence of
maturation/differentiation markers on DC2 cells that they are
unresponsive to inflammatory stimuli and, therefore, functionally
inactive at the cutaneous site. However, this interpretation is at odds
with the findings that DC2 cells within the skin down-regulated CD86
expression and markedly up-regulated expression of CCR5, a principal
receptor for inflammatory chemokines (82). Whereas a
variety of surface markers and cytokines appear to be useful for
assessing activation of DC1 cells (47), evaluation of the
responsiveness of DC2 cells to cytokines and PAMPs appears to be
critically dependent upon an awareness of the more limited biosynthetic
repertoire of these cells under stimulatory conditions. Recently, Cella
and coworkers proposed that DC2 cells maintain the efficiency of T cell
responses and protect against the cytopathic effects of virus by
producing large amounts of IFN- within inflamed lymph nodes
(49). In light of in vitro findings that
lipoprotein-mediated responses are proapoptotic (22), it
is tempting to speculate that DC2 cells might fulfill a similar
protective role during syphilitic infection.
The epidemiologic relationship between genital ulceration due to
syphilis and the acquisition of HIV-1 infection was recognized early in
the AIDS epidemic (83). Although initially attributed to
the disruption of epithelial barriers, it soon was appreciated that
genital ulcers comprise a unique inflammatory niche well endowed with
HIV-1 target cells (1, 54). Herein we used our human skin
model to extend prior in vitro experiments demonstrating that
treponemal lipoproteins/lipopeptides induced reciprocal changes in the
expression of CCR5 and CXCR4 on PB monocytes (20). We
found, in fact, that these proinflammatory agonists have the capacity
to establish a microenvironment highly enriched for mononuclear cells,
particularly macrophages and DCs, expressing the CCR5 coreceptor for
sexually transmitted, M-tropic strains of HIV-1. These observations
also have broad implications for our understanding of the ontogeny of
the inflammatory response in early syphilis. During infection, diverse
immune cells responding to the presence of these chemoattractants would
be trapped in the inflammatory focus until the local concentration of
lipoproteins is diminished by clearance of organisms, at which point
they would be free to respond to other chemoattractants, directing
their return to the circulation or migration to skin-draining lymph
nodes. This scenario, interestingly, is at odds with the current
paradigm for DC migration stating that CC chemokine receptors,
including CCR5, are essential for the homing of DCs to inflamed
tissues, but are rapidly down-regulated once within sites of
inflammation (82, 84). It should be noted that this
paradigm is derived largely from in vitro studies with LPS and
proinflammatory cytokines (e.g., IL- 1
and TNF-) (85, 86), whereas our findings reflect the complex immunomodulatory
networks and cell-to-cell interactions occurring in vivo. Moreover, LPS
and lipoproteins/lipopeptides engender divergent effects on CCR5/CXCR4
expression (20), with the former down-regulating and the
latter up-regulating the expression of CCR5. These converse effects on
CCR5 expression reflect the use by these two PAMPs of different TLRs
for stimulating innate immune cells (T.J.S. and J.D.R., unpublished
observations) (22, 23, 24, 25, 26, 87). Because Gram-positive
bacterial skin pathogens, such as spirochetes, activate cells via TLR2
(24, 27, 88), our findings also may pertain to other
clinically relevant cutaneous infections.
Histochemical studies have established that primary and secondary syphilitic lesions contain highly similar cellular infiltrates consisting predominantly of lymphocytes and macrophages (1, 4, 5, 6, 7, 8). Moreover, preliminary evidence from application of the suction blister technique to syphilis patients demonstrates that, similar to lipopeptide injection sites, secondary skin lesions are enriched for macrophages, CLA+ T cells, and DCs (data not shown). These findings lead us to propose that the similarities between primary and secondary syphilis lesions reflect, to a large extent, the stereotypical nature of the proinflammatory response to treponemal lipoproteins. If so, it is tempting to speculate that the character of the subsequent adaptive response would be determined by whether the memory/effector T lymphocytes elicited as part of this process possess immunologic memory for T. pallidum Ags. Early in the development of a chancre, a primary immune response would ensue because the infiltrating memory/effector T cells are not sensitized to treponemal Ags. However, infiltrating DCs will take up Ag for presentation to naive T cells within regional lymph nodes. Trafficking of these neo-sensitized cells back to the genital ulcer would then accelerate bacterial clearance and subsequent healing of the primary lesion. In secondary syphilis lesions, the presence of mature DCs, as well as other APCs (i.e., activated macrophages), coupled with the influx of T. pallidum-sensitized T cells produced during the primary stage of infection, would foster a robust and rapid local secondary immune response, signaling a potential turning point in the host’s efforts to contain the pathogen and establish latency.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Justin D. Radolf, Center for Microbial Pathogenesis, University of Connecticut Health Center, Farmington, CT 06030-3710.
3 Abbreviations used in this paper: TLR, Toll-like receptor; CLA, cutaneous lymphocyte Ag; CXCR, CXC chemokine receptor; DC, dendritic cell; FA, fluorescence assay; PAMP, pathogen-associated molecular pattern; PB, peripheral blood.
Received for publication October 3, 2000. Accepted for publication January 3, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 H. Crandall, D. M. Dunn, Y. Ma, R. M. Wooten, J. F. Zachary, J. H. Weis, R. B. Weiss, and J. J. Weis Gene Expression Profiling Reveals Unique Pathways Associated with Differential Severity of Lyme Arthritis J. Immunol., December 1, 2006; 177(11): 7930 - 7942. [Abstract] [Full Text] [PDF]
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 R. E. LaFond and S. A. Lukehart Biological Basis for Syphilis Clin. Microbiol. Rev., January 1, 2006; 19(1): 29 - 49. [Abstract] [Full Text] [PDF]
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 J. C. Salazar, C. D. Pope, M. W. Moore, J. Pope, T. G. Kiely, and J. D. Radolf Lipoprotein-Dependent and -Independent Immune Responses to Spirochetal Infection Clin. Vaccine Immunol., August 1, 2005; 12(8): 949 - 958. [Abstract] [Full Text] [PDF]
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M. R.-E.-I. Benhnia, D. Wroblewski, M. N. Akhtar, R. A. Patel, W. Lavezzi, S. C. Gangloff, S. M. Goyert, M. J. Caimano, J. D. Radolf, and T. J. Sellati Signaling through CD14 Attenuates the Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease J. Immunol., February 1, 2005; 174(3): 1539 - 1548. [Abstract] [Full Text] [PDF]
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B. A. Zabel, A. M. Silverio, and E. C. Butcher Chemokine-Like Receptor 1 Expression and Chemerin-Directed Chemotaxis Distinguish Plasmacytoid from Myeloid Dendritic Cells in Human Blood J. Immunol., January 1, 2005; 174(1): 244 - 251. [Abstract] [Full Text] [PDF]
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 N. J. Megjugorac, H. A. Young, S. B. Amrute, S. L. Olshalsky, and P. Fitzgerald-Bocarsly Virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of T and NK cells J. Leukoc. Biol., March 1, 2004; 75(3): 504 - 514. [Abstract] [Full Text] [PDF]
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J. C. Salazar, C. D. Pope, T. J. Sellati, H. M. Feder Jr, T. G. Kiely, K. R. Dardick, R. L. Buckman, M. W. Moore, M. J. Caimano, J. G. Pope, et al. Coevolution of Markers of Innate and Adaptive Immunity in Skin and Peripheral Blood of Patients with Erythema Migrans J. Immunol., September 1, 2003; 171(5): 2660 - 2670. [Abstract] [Full Text] [PDF]
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D. M. Koelle and L. Corey Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research Clin. Microbiol. Rev., January 1, 2003; 16(1): 96 - 113. [Abstract] [Full Text] [PDF]
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T. L. Humphreys, C. T. Schnizlein-Bick, B. P. Katz, L. A. Baldridge, A. F. Hood, R. A. Hromas, and S. M. Spinola Evolution of the Cutaneous Immune Response to Experimental Haemophilus ducreyi Infection and Its Relevance to HIV-1 Acquisition J. Immunol., December 1, 2002; 169(11): 6316 - 6323. [Abstract] [Full Text] [PDF]
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