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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-04-1019.
PHAGOCYTES
From the Center for Comparative Medicine, Schools of
Medicine and Veterinary Medicine; Department of Biomedical Engineering;
and Department of Veterinary Medicine: Pathology, Microbiology and
Immunology, University of California, Davis; and Section of
Atherosclerosis and Leukocyte Biology, Departments of Medicine and
Pediatrics, Baylor College of Medicine, Houston, TX.
Tick saliva contains anti-inflammatory and immunosuppressive
substances that facilitate blood feeding and enhance tick-vectored pathogen transmission, including Anaplasma phagocytophila,
an etiologic agent of granulocytic ehrlichiosis. As such, inflammation at a tick-feeding site is strikingly different than that typically observed at other sites of inflammation. Up-regulation of CD11b/CD18 occurs in host granulocytes following interaction or infection with
A phagocytophila, and the absence of CD11b/CD18
results in early increases in bacteremia. We hypothesized that
Anaplasma phagocytophila, formerly named
the agent of human granulocytic ehrlichiosis (HGE),1 is an
obligate intracellular granulocytotropic bacterium that relies on a
short-lived, terminally differentiated, powerful antimicrobial cell for
survival and dissemination.2 A phagocytophila
is vectored by ticks in the Ixodes persulcatus complex3,4 and is 1 of 2 recognized pathogens to cause
HGE.5 Laboratory mice serve as useful tools for
investigation of in vivo pathogenesis and kinetics of A
phagocytophila infection and are uniquely suited to study the
natural route of infection and transmission at the host-vector
interface.3,4,6,7 In addition, the use of genetically
engineered mice with precise genetic defects, including deletions of
leukocyte adhesion molecules, allows detailed investigation of
infection kinetics, leukocyte function and migration, and pathogen
acquisition. Infection of mice with A phagocytophila results
in increased surface expression of the The kinetics of neutrophil movement in blood, extravasation, and
trafficking to the dermis are multistep navigational processes reliant
on adhesion molecules found on neutrophils, endothelial cells,
connective tissue cells, and extracellular matrix proteins. Two A variety of individual chemotactic and/or activation stimuli have been
used in the study of dermal leukocyte migration both in vivo and in
vitro. However, specific evaluation of leukocyte migration to
tick-feeding sites has received little attention. Proteins within tick
saliva inhibit activation of the alternative complement
pathway,20,21 interferon (IFN- Mice
A phagocytophila
Ticks Adult Ixodes scapularis ticks were collected from the field in southern Connecticut and generously provided by Dr Durland Fish (Yale University, New Haven, CT). Adult ticks produced larvae that were fed on uninfected C3H/HeN mice and allowed to molt and harden into nymphs as previously described.7 Ten percent of the molted nymph pool was tested by PCR and found to be negative for Borrelia burgdorferi and A phagocytophila.In vivo experimental design Mice were inoculated with infected (4 × 105 to 1 × 106 A phagocytophila p44 DNA copy number per mouse, depending on experiment) or uninfected blood intraperitoneally on day 5. On day 5 after inoculation (experimental
day 0), infected and control mice were anesthetized with 0.15 to 0.20 mL ketamine/xylazine intraperitoneally (10 mg ketamine per 1 mg
xylazine per milliliter, Vetamine, Schering-Plough Animal Health,
Union, NJ; TranquiVed, Vedco, St Joseph, MO), and each mouse was
infested with 5 uninfected nymphal ticks. Nymphs were placed in small
shaved patches on the cranial to middorsal midline. At the same time,
one suture (3-0 silk, Ethilon) was placed through the skin in a
small shaved area above the tail head, distant from the nymphs. Suture
served as a nonspecific, alternate inflammatory source and as a
positive control, as described.7 Groups of 3 to 6 uninfected and infected mice of each strain (excluding CD18 /) were killed with CO2 at 0, 24, 48, and 72 hours after tick infestation (corresponding to days 5, 6, 7, and
8 after initial infection). Previous experiments have shown that marked
bacterial amplification in blood occurs by day 3 to 4 after infection
and bacteremia is stable by day 5. In addition, this interval
represents the preimmune stage of disease, avoiding the complication of
immune-mediated pathogen clearance.
At necropsy, blood from each mouse was obtained for quantitative PCR, complete blood cell counts, and differential leukocyte counts. Skin samples were collected from multiple sites including tick-feeding site(s), distant nontick sites, and suture sites using 2-mm sterile dermal biopsy punches (Miltex Instrument, Bethpage, NY) to ensure comparably sized tissue sections for quantitative PCR. Skin from tick, nontick, and suture sites was also collected for histopathology. The location of ticks was noted, and all ticks were individually collected for quantitative PCR except for ticks attached to skin sections submitted for histopathology, which were used to direct and facilitate sectioning. For histopathology, sections of skin were fixed in 10% formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin using standard techniques. All slides were blindly examined by one of the authors (H.E.V.D.) to assess the degree and type of inflammatory cell infiltrate and presence or absence of transmigrating neutrophils as well as other significant inflammatory changes including hemorrhage and edema. DNA extraction and quantitative PCR DNA extraction. DNA was extracted from 50 µL blood, individual skin samples, and individual ticks obtained at necropsy using DNeasy tissue kit according to the manufacturer's instruction for tissues and insects (QIAGEN, Valencia, CA). Ticks were crushed in liquid nitrogen with a plastic grinder, and powder was used for consequent DNA extraction. The copy number of p44 A phagocytophila target gene was expressed per tick, per skin sample, and per milliliter of blood. TaqMan probe, primers, reaction mix, and thermal cycling. Three oligonucleotides, 2 primers, and 1 probe for the target gene A phagocytophila p44 (GenBank accession no. AF037599) were used under the conditions previously described.8 DNA amplification, data acquisition, and data analysis were performed in an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA), and quantification of the amount of A phagocytophila p44 gene in each sample was accomplished by measuring cycle threshold (CT) and using an absolute standard curve (from a plasmid standard) as previously described.30 A Sequence Detection System (version 1.6 software) was used to analyze data. Results were exported to a Microsoft Excel worksheet for statistical analysis. Plasma IFN-  levels were assayed using an OptEIA
enzyme-linked immunosorbent assay (ELISA) kit according to
manufacturer's instructions (Pharmingen, San Diego, CA). In brief,
96-well plates were coated with 100 µL antimouse IFN- capture
antibody (diluted at 1:2000 in coating buffer) and incubated overnight
at 4°C. Wells were washed and blocked with phosphate-buffered
saline (PBS)/10% fetal bovine serum (FBS) for 1 hour. A
standard curve was created using recombinant mouse IFN- diluted with
PBS/10% FBS (1000 pg/mL to 31.3 pg/mL); 100 µL of each standard,
sample, or controls was pipetted into each well and incubated for 2 hours at room temperature (samples were run in duplicate at 1:4 and 1:8
dilutions). Plates were washed, and 100 µL biotinylated antimouse
IFN- (diluted 1:250) was added to each well. After 1 hour at room
temperature, plates were washed and 100 µL avidin-horseradish
peroxidase conjugate was added. Also, 100 µL of the substrate
(tetramethylbenzidine and hydrogen peroxide) was added and permitted to
incubate for 30 minutes. Optical absorbance was read at 450 nm on an
automated ELISA plate reader.
Statistical analysis Statistical comparisons between 2 groups of mice or time periods were made using a Student t test. Multiple comparison analyses were made using a 1-way analysis of variance (ANOVA) followed by a least squares difference post hoc test (SPSS, version 6.1 for Windows, SPSS, Chicago, IL). Calculated P values less than .05 were considered significant.
Tick feeding results in increased A phagocytophila DNA
in blood with kinetics influenced by / and
CD11b/ mice had significantly higher p44 DNA
in their blood compared with B6 wild-type controls
(P = .024 and .038, respectively). At 24 hours after tick
infestation, there was a significant and marked increase in p44
DNA in the blood of all mouse strains compared with time 0 (P < .05 for all strains). This increase, however, was
3-fold greater for CD11a/ and CD11b/
mice compared with B6 wild-type controls (P = .003 and
.007, respectively).
Plasma IFN- The kinetics of A phagocytophila p44 DNA copy number in
blood changed over the course of tick feeding. In B6 mice, a
significant increase in p44 DNA began by 24 hours after tick
infestation, continued through 48 hours, and returned to baseline
levels at 72 hours. In CD11b A peripheral neutrophilia developed in infected mice, but not in
uninfected control mice, 24 hours after tick infestation (Table
1). Compared with time 0, infected mice
from all strains had a significantly higher neutrophil count at 24 hours after tick infestation than their baseline neutrophil counts
(P < .05 for all comparisons). This neutrophilia was also
noted in CD18
To distinguish between the contribution of increased pathogen
replication within an individual neutrophil and the contribution of an
increase in the population of susceptible cells to total blood pathogen
burden, p44 DNA copy number was normalized by granulocyte number per milliliter of blood. This ratio is depicted in Figure 1B. At
time 0, the significant increase of p44 DNA in the blood of
CD11a In summary, tick feeding resulted in a marked amplification of
p44 DNA in the blood of all infected mice. The kinetics of this increase differed between mice. The amplification could be partially explained by an increase in susceptible host cells
(neutrophilia) in all mice. CD11b Neutrophil-pathogen accumulation at dermal sites of suture and tick feeding Suture elicited significantly more neutrophilic inflammation and pathogen accumulation than tick feeding based on histopathology and quantitative PCR of skin biopsies (Figures 2-3; Table 2). As such, these disparate tissue insults provided 2 extremes of inflammatory stimuli. In most mice, histopathologic changes within 24 hours of suture placement included marked diffuse neutrophilic, occasionally necrotizing, inflammation in the superficial dermis. This inflammation was directly associated with the suture and probably represented recruitment of local neutrophils to trauma and local "injection" of skin-associated bacterial flora into the skin (Figure 2A). Remarkably, there was an absence of neutrophilic inflammation in response to suture in CD18/ mice (Figure 2B). In addition, marginated and
transmigrating neutrophils, distant from the inciting suture, were
noted in B6 mice but were essentially absent in CD11a/,
CD11b/, and CD18/ mice. In sharp
contrast, histopathologic changes associated with 24 to 48 hours of
tick feeding included mild to moderate, diffuse, primarily
lymphoplasmacytic inflammation in the superficial dermis, regardless of mouse strain. The tick attachment site was characterized by a cement cone, secreted by the tick, that appeared in tissue sections as a homogeneous eosinophilic matrix (Figure
2C-D).31,32 After 72 hours, features associated with tick
feeding included foci of moderate to marked hemorrhage with swollen,
plump endothelial cells, edema, and vascular congestion (Figure 2E-F).
These vascular changes were not noted at suture sites, even in the face
of occasionally necrotizing, marked neutrophilic inflammation.
In the absence of stimuli, A phagocytophila p44 DNA copy number in the skin of infected mice was generally low, in agreement with previous studies.30 Two methods were used to compare the efficiency of leukocyte-pathogen migration to sites of dermal inflammation. The first simply compared total p44 DNA copy number per skin sample (Figure 3). The second method normalized for the contribution of increased total blood p44 DNA in knock-out mice by computing the ratio of copy number in tissue sample to that in blood multiplied by 100 (Table 2). This ratio permitted evaluation of leukocyte trafficking to the dermis while compensating for the marked increase in p44 DNA copy number in blood of knock-out mice. A phagocytophila p44 DNA was significantly
increased in skin biopsies from both suture and tick-feeding sites
compared with adjacent noninflamed sites in all 3 mouse strains at all
time periods (Figure 3; P < .05 for all comparisons).
However, amplification of p44 DNA was 100 to 150 times
greater at suture sites than at tick-feeding sites on the same mouse
(note y-axis, Figure 3A-B). At 24 hours after infestation, no
significant differences between mouse strains were noted in total
p44 DNA from either tick feeding or suture skin. At 48 hours, tick skin from CD11a There was a significantly higher ratio of p44 DNA at suture
sites compared with tick-feeding sites at all time periods and for all
mouse strains (Table 2). Data summarized in Table 2 highlight the trend
of high relative pathogen DNA at suture sites that progressively
decreased at tick-feeding sites and in feeding ticks. At 24 hours,
knock-out mice showed a decreased ratio of p44 DNA at both
suture and tick sites compared with wild-type mice. This may reflect a
delay or deficit in neutrophil extravasation or trafficking. However,
by 48 hours, all mice showed essentially comparable ratios of p44
DNA at sites of dermal inflammation. With the exception of
CD11b In summary, suture and tick stimuli elicit different types of dermal inflammation. Although both stimuli resulted in increased dermal A phagocytophila p44 DNA compared with noninflamed skin, suture elicited greater neutrophil-pathogen accumulation. At 24 hours, all knock-out mice had a higher absolute amount of p44 DNA at tick-feeding sites compared with wild-type mice. This likely reflected the increase in total blood pathogen burden, because the ratio of p44 DNA in skin compared with blood was actually lower in knock-out mice than in wild-type mice. This may represent an early deficit in neutrophil extravasation or trafficking. By 48 to 72 hours, the ratio of pathogen in blood compared with skin normalized, likely reflecting hemorrhage at tick-feeding sites rather than more effective neutrophil trafficking. Efficient pathogen transmission from infected mice to I
scapularis nymphal ticks is independent of After 24 hours of feeding, more than 75% of the ticks were positive
for A phagocytophila p44 DNA, with no significant
difference between mouse strains in the overall percent of positive
ticks. Figure 4 depicts the average copy
number of p44 DNA amplified from ticks feeding on the backs
of mice over time. Despite a significantly higher infection burden in
the blood of CD11a
As before, a ratio was created by dividing A phagocytophila
p44 DNA copy number per tick by p44 DNA copy
number in blood to compensate for variations in bacteremia. This ratio
permitted determination of relative transmission efficiency as a
function of the presence or absence of
Inflammation stimulates the release or synthesis of
prostaglandins, leukotrienes, and platelet-activating factor, which, in turn, have profound effects on vascular tone, blood flow, and leukocyte
diapedesis. Experimentally, leukocyte diapedesis and migration are
often studied in the context of a single stimulus to distinguish
between responses to individual inflammatory mediators. However, tick
saliva is a unique stimulus, because it is replete with substances that
modulate, and generally down-regulate, inflammatory and hemostasis
cascades as well as alter leukocyte functions
(Wikel33 offers a complete review). The purpose
of this study was to extend the previous observation of neutrophil
activation and In the present study, neutrophil All infected mice developed a peripheral neutrophilia 24 hours after
tick infestation, concurrent with the marked increase in pathogen
burden in blood. We have previously noted the induction of neutrophilia
concurrent with A phagocytophila expansion in murine blood
(D.L.B., unpublished observations, 2001). Likely considerations for this infection-dependent neutrophilia include the
induction of IL-8 (or its murine homolog) by host granulocytes or
delayed apoptosis of circulating neutrophils.36-38A
significant and relatively sustained neutrophilia in
CD11a In addition to a moderate neutrophilia, neutrophils from
CD11b A phagocytophila is not a dermatotropic pathogen and cannot be readily amplified in high numbers from skin biopsies during active infection.30 For a pathogen that relies on hematogenous acquisition, it is not surprising that skin sites associated with a feeding tick, or other inflammatory stimuli, contain significantly greater Anaplasma DNA than adjacent nontick sites. These data are consistent with previously published observations on the acquisition dynamics of A phagocytophila at the host-vector interface that demonstrated peak skin infection at 24 hours, diminishing by 48 and 72 hours after tick infestation.7 Suture, as a nonspecific inflammatory source, readily recruits neutrophils and Anaplasma DNA from the blood into dermal tissue.7 Our findings concur and suggest that A phagocytophila-infected neutrophils are recruited to inflammatory stimuli in response to generic chemotactic gradients and not only specifically in response to elements in tick saliva. Although suture elicited up to 150-fold more neutrophilic inflammation than feeding ticks (Figures 2 and 3), the inflammation was not sustained. One probable reason for the rapid fall in leukocyte-pathogen recruitment is that a number of mice (more than 50%) were able to groom and remove the suture. Thus, the physical stimulus was frequently absent after 24 to 48 hours, whereas tick feeding provided stimulation throughout the experiment. In addition, acute, neutrophilic inflammation secondary to a single stimulus generally peaks by 24 hours and recedes. Regardless, suture sites remained visible and palpable even in mice where the suture had been removed, and comparisons between sites where suture had been removed versus sites where suture remained revealed no significant differences in quantitative Anaplasma DNA copy number. Conversely, tick feeding evoked a very different type and degree of inflammatory response. The cellular response to tick feeding varies with host species, tick species, and number of tick exposures. Sequential histopathology of Rhipicephalus sanguineus tick-feeding sites in dogs and guinea pigs revealed variability in type of cellular infiltrate, primarily neutrophilic in dogs, with a peak at 24 hours of infestation, and mononuclear/mastocytic in guinea pigs.31 In the current study, 24 hours of tick feeding induced focal, mild to moderate diffuse, lymphocytic dermatitis with minimal hemorrhage (Figure 2). These findings are consistent with previous reports of lymphocytic (CD4+) inflammation in response to tick feeding in BALB/c mice.39 Leukocyte trafficking may be a primary determinant of early
leukocyte-pathogen recruitment to tick-feeding sites, when a feeding cavity has not been fully established, and in peripheral tissues not
directly associated with feeding (Figure 2).31,40 This trafficking is complex and may rely on modulated expression of the In these studies, we evaluated the influence of
The authors gratefully acknowledge the technical assistance of Kim Freet, Bob Munn, Judy Walls, and Amy Smith.
Submitted April 9, 2002; accepted November 25, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002- 04-1019.
Supported by grants AI-41440 and RR-07038 (S.W.B.), AI-47294 (S.I.S.), and R01-HL62243-01 (C.M.B.) from the National Institutes of Health (NIH). S.I.S. and C.M.B. are Established Investigators of the American Heart Association. D.L.B. was supported by NIH training grant T32 RR-07038.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen W. Barthold, Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, CA 95616; e-mail: swbarthold{at}ucdavis.edu.
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2 integrins in kinetics of bacteremia,
extravasation, and tick acquisition of Anaplasma
phagocytophila in mice
Top
Abstract
Introduction
2 integrin-dependent infection kinetics and leukocyte
extravasation are important determinants of neutrophil
trafficking to, and pathogen acquisition at, tick-feeding sites.
A phagocytophila infection kinetics were evaluated in
CD11a/CD18, CD11b/CD18, and CD18 knock-out mice using quantitative
polymerase chain reaction (PCR) of blood, ticks, and skin biopsies in
conjunction with histopathology. A marked increase in the rate of
A phagocytophila infection of neutrophils and pathogen
burden in blood followed tick feeding. Infection kinetics were modified
by 
, -
