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,,
,
Departments of
*
Medicine,
Immunology, and
Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, CO 80262; and Departments of
Medicine and
¶ Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell subsets. Studies of peripheral blood T cells before and after
patch testing provided evidence for mobilization of large numbers of
pathogenic beryllium-reactive T cells into the circulating pool. These
studies using skin patch testing provide new insight into the dynamics
of T cell influx and mobilization during granulomatous
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Considerable evidence suggests that CD4+ T cells are important in the immune response to beryllium and in the immunopathogenesis of CBD (11, 16, 17, 18, 19). For example, the development of granulomatous inflammation is associated with the accumulation of CD4+ T cells in the bronchoalveolar lavage (BAL). In a number of individuals, these T cells included oligoclonal T cell expansions with particular TCRs specific for CBD (19). CD4+ T cells in the BAL of CBD patients have been shown to proliferate and release Th1 cytokines in vitro in the presence of APCs expressing class II MHC molecules (14, 15, 17). It is currently unknown whether the T cells present in BAL fluid reflect the same T cells that drive granulomatous inflammation in the tissue. Lung tissue is relatively inaccessible to repeated sampling or to large samples, as are many organs affected by immune-mediated disease. In addition, the CD4+ T cells in BAL have been studied only after many years of disease, after granulomas are well established. Finally, CD4+ T cell function has been assessed primarily by in vitro testing. An assay system that analyzed the in vivo T cell-dependent process in the development of granulomatous diseases would likely add important new insight into the disease process.
The immune basis of CBD was first suggested by the demonstration of a delayed-type hypersensitivity response in beryllium-exposed subjects following skin patch testing with beryllium salts (20, 21). However, the use of beryllium patch testing as a diagnostic tool decreased in popularity because of the potential risk of inducing sensitization and the theoretical risk that the underlying disease would be exacerbated (22). Recently, patch testing has been reinvestigated as a diagnostic tool (23, 24, 25). In 14 subjects with CBD, positive patch tests were associated with no detectable adverse effects (25). In the present study, we used beryllium patch testing in CBD patients to compare the repertoire of T cells infiltrating skin with BAL T cells in the same individual. We noted that the CD4+ T cells infiltrating skin in response to beryllium varied depending on the timing and pathology. Early T cell infiltrates in the skin, before granuloma formation, expressed a different set of TCRs. In contrast, later in the course of granuloma formation, a significant fraction of skin T cells matched those present in BAL and expressed beryllium-reactive TCRs previously identified as specific for CBD. These T cell specificities were also detected for the first time in the circulating pool. These studies demonstrate an in vivo model of granuloma formation in humans with mobilization and influx of pathogenic CD4+ T cells similar to that which occurs in the internal organ primarily affected by this disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diagnosis of CBD was established using previously defined
criteria (11, 26), including a history of beryllium
exposure, the presence of granulomatous inflammation on lung biopsy,
and a positive proliferative response of BAL T cells to beryllium
sulfate (BeSO4) in vitro. We studied three CBD
patients in whom CD4+ T cell expansions were
previously identified in the BAL (two with TCR V3- and one with TCR
V14-expressing expansions) (18). CBD patients 1 and 3
were being treated with corticosteroids at the time of enrollment in
this study. Informed consent was obtained from each CBD patient, and
the protocol was approved by the Human Subject Institutional Review
Boards at the University of Colorado Health Sciences Center and
National Jewish Medical and Research Center (Denver, CO).
BeSO4 patch testing of CBD patients
Patch testing was performed using a modified version of the
methods of the North American Dermatitis Group (27). One
percent aqueous BeSO4 in Finn chambers was
applied to normal unabraded skin on the back for 48 h. The patch
test was interpreted at 96 h as follows: 1+, weak (nonvesicular)
positive reaction; 2+, strong (vesicular) positive reaction; 3+,
extreme (bullous) positive reaction. A 3- or 4-mm punch biopsy was
performed at abnormal patch test sites at 96 h and after 
2–5
wk. A patch test with normal saline was also performed at day 0 and
biopsied at 96 h. A biopsy of normal skin was performed at day 0.
Skin punch biopsy specimens were placed in cell lysis solution and
homogenized with a glass/Teflon homogenizer (VWR Scientific
Products, Aurora, CA) and stored at -70°C until processed for
analysis.
Immunohistochemistry of BeSO4 skin patch tests
A second punch biopsy was collected from each subject at the designated time points, frozen, and cut into 5-µm sections. Abs directed against CD3, CD4 (both from BD Biosciences, San Jose, CA), and CD8 (monoclonal IgG1; DAKO, Carpinteria, CA) were used for immunohistochemistry. Avidin-biotin complex peroxidase methods were used for staining based on VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, then blocked with 1.5% normal horse serum for 30 min. Sections were incubated for 60 min with the primary Ab diluted in 1.5% normal horse serum, washed in PBS, and incubated for 30 min in biotinylated horse anti-mouse IgG Ab. Following washing, sections were incubated for 30 min with HRP-conjugated avidin-biotinylated complex, washed again with PBS, then developed with 3'-diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin. All samples were also stained using H&E.
Isolation and analysis of TCR expression of peripheral blood and BAL CD4+ T cells
Mononuclear cells were isolated from heparinized blood by Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient separation and from BAL as previously described (28). Peripheral blood cells from CBD patients were stained with FITC-labeled anti-CD4 mAb (BD Biosciences), and CD4+ T cells were sorted using a MoFlo cell sorter (Cytomation, Fort Collins, CO). Sorting of BAL cells from CBD patients for CD4+ T cells was not performed, due to the >80% predominance of CD4+ T cells in this compartment.
Mononuclear cells were analyzed by two-color immunofluorescence for CD4
and TCR V region gene expression using mAbs directed to 11 different
TCR V receptors and one TCR V
receptor as previously described
(18, 29). Anti-TCR mAbs were biotinylated and
streptavidin-PE (Fisher Biotech, Pittsburgh, PA) was used as a
second-step reagent for TCR staining. Cells were also double-stained
with FITC-labeled CD3, CD4, and CD8 (all from BD Biosciences). The
lymphocyte population was identified using forward and 90° light
scatter patterns, and fluorescence intensity was analyzed using a
FACScan cytometer (BD Biosciences) as previously described.
Analysis of TCRBV gene expression in peripheral blood, BAL, and skin biopsies
Total RNA was isolated using an acid guanidinium-phenol
chloroform method, and cDNA was synthesized using 2 µg RNA per
20-µl reaction. cDNA synthesis reagents included reverse
transcriptase (SuperScript RT; Life Technologies, Grand Island, NY),
RNase inhibitor (Promega, Madison, WI), and dNTPs and random hexamers
(both from Amersham Pharmacia Biotech). One microliter of the cDNA
reaction was added per 50 µl PCR mixture. PCR amplification
(AmpliTaq; PerkinElmer, Branchburg, NJ) was performed for 35 cycles,
and both 5' and 3' oligonucleotide primers were present at a
concentration of 0.3 µM. Sequences of the 5' TCR -chain gene
(TCRB)V3 primer, 5' TCRBV14 primer, 5' TCRBV2 primer, and the 3' CB
primer are 5'-GTCTCTAGAGAGAAGAAGGAGCGC-3',
5'-GTCTCTCGAAAAGAGAAGAGGAAT-3', 5'-GCAACTTCCAATGAGGGCTCC-3',
and 5'-TTCTGATGGCTCAAACAC-3', respectively. The PCR products were
ligated into the pCR II TA cloning vector (Invitrogen, San Diego, CA),
and the ligation products were transformed into Epicurian Coli XL-1
Blue supercompetent Escherichia coli cells (Stratagene, La
Jolla, CA). Colonies containing inserts were randomly selected for
nucleotide sequencing. Cycle sequencing was performed using M13 reverse
(5'-CAGGAAACAGCTATGAC-3') and/or M13 forward
(5'-CCCAGTCACGACGTTGTAAAACG-3') sequencing primers and an automated ABI
377 sequencer (PE Applied Biosystems, Foster City, CA).
Lymphocyte proliferation assay
Proliferation assays were performed using PBMCs (2.5 x 105 cells/well) cultured in 96-well flat-bottom microtiter plates in the presence of 1 µg/ml PHA for 48 h or BeSO4 at concentrations ranging from 1 x 10-4 to 1 x 10-6 M for 96 h. The wells were then pulsed with 1 µCi of [3H]thymidine for an additional 18 h, and incorporation of radioactivity was determined by beta emission spectroscopy. Proliferation assays were performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously identified a subset of CBD patients with
CD4+ T cell subset expansions in the BAL compared
with blood (18, 19). Three of these individuals were
selected to undergo BeSO4 patch testing in the
current study and were restudied for alterations in TCR V region
expression. The percentage of CD4+ T cells in the
BAL and blood expressing particular V regions for patients 1–3 was
determined by immunofluorescence staining and cytofluorographic
analysis (Fig. 1
). Patient 1 showed an
increased percentage of V3+ and
V13.1+ T cells in the BAL compared with the
blood population (for V3, 12.3 vs 6.1%, respectively). In patient
2, 9.3% of the CD4+ T cells in the BAL expressed
V3 as compared with 4.6% in the blood. Patient 3 demonstrated a
V14 expansion with 10.8% of the CD4+ T cells
in the BAL expressing V14 compared with 3% in the blood.
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At 96 h after application of 1% BeSO4, all three individuals developed a strongly positive (2+) patch test reaction characterized by erythema, induration, and vesicles. No individual developed a positive reaction to normal saline. A separate study from this laboratory showed that control subjects with contact dermatitis and no history of beryllium exposure fail to develop a positive skin reaction following BeSO4 patch testing (25). Thus, patch testing of control subjects was not repeated in this study.
Skin biopsies obtained at 96 h after patch testing showed mild to
moderate spongiosis involving the lower layers of the epidermis and
focal edema of the papillary dermis (Fig. 2A). In contrast to the skin
of healthy individuals, which usually harbors only a small number of
lymphocytes, these biopsies showed a significant lymphocytic infiltrate
in a perivascular distribution. There was no evidence of granulomatous
inflammation at this early time point. Immunohistochemistry of the
biopsy showed that the infiltrating lymphocytes expressed CD3 and CD4
(data not shown). Few CD8+ T cells were
present.
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2–5
wk. As shown in Fig. 2
B, the spongiosis of the epidermis and
the edema of the papillary dermis were resolved. Fig. 2C
shows a poorly formed noncaseating granuloma. Staining of these lesions
with anti-CD68 showed clusters of histiocytes and confirmed their
granulomatous nature (data not shown). Similar to the findings at
96 h, the lymphocytic infiltrate was primarily composed of
CD3+ cells (Fig. 2D) that expressed
CD4 (Fig. 2E). Few, if any, CD8+ T
cells were present. Analysis of expressed TCRB genes in CBD patients
We focused on the TCR V regions expressed by expanded
CD4+ T cell subsets in the BAL. Selection for
skin CD4+ T cells was not performed due to the
rarity of CD8+ T cells in the skin biopsies and
the difficulty of obtaining adequate numbers of lymphocytes in a 3-mm
punch biopsy specimen. BAL cells from CBD patients were also not sorted
for CD4+ T cells due to the great predominance of
CD4+ T cells in this compartment. However, we
sorted CD4+ T cells from the blood of individual
patients from which RNA and cDNA were prepared. TCRBV3 fragments from
patients 1 and 2 and TCRBV14 fragments from patient 3 were amplified,
cloned in bacteria, and sequenced. Fig. 3
shows junctional region sequences from patient 1 that were found at
least two times in normal skin and in skin 96 h after either
normal saline or BeSO4 patch testing. Each of
these biopsies contained oligoclonal T cell populations, with a few
clonal expansions approaching 20–30% of the
V3+ subset. We detected repeated sequences of
individual T cell clones in control samples (normal skin and skin
following normal saline patch testing) and in the 96-h skin biopsy
following BeSO4 patch testing. However, none of
these apparently expanded clonal populations in skin were identified in
BAL, blood, or later samples of skin (Fig. 3), and none of the TCRs
expressed by BAL expansions were detected in these skin samples (see
below). In addition, no TCR sequence similarities were present among
these different skin samples, e.g., normal skin (day 0) vs after saline
or beryllium patch testing. The presence of clonal populations in
normal skin and skin following normal saline patch testing in the face
of no inflammation and few infiltrating lymphocytes could have been due
to the very small amounts of TCR mRNA initially present and subsequent
PCR amplification. Clonal expansions (i.e., two or more repeated
sequences from the same T cell clone) were completely absent from the
peripheral blood V3+CD4+
subset of patient 1.
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). For example, in patient 1, one TCRBV3
sequence (BV3-QGD-BJ1S5) accounted for 6 of 64 BV3 sequences (9.4%) in
BAL and 2 of 39 (5.1%) in the skin (Fig. 4). This and other TCRBV3
sequences present in skin and/or BAL were noted to have a TCR
complementarity-determining region (CDR)3 motif characteristic
of pathogenic beryllium-reactive cells in CBD, and some sequences were
identical to those previously described (19). This CDR3
motif includes an invariant aspartate (D) at position 96 of the
-chain and a length of 8 amino acids. One of the larger BAL clonal
populations, BV3-PGGGLG-BJ2S4, accounted for 16% of the BAL TCRBV3
repertoire and 5.1% of the skin TCRBV3 sequences. Overall, 21% of the
skin TCRBV3 repertoire matched sequences present in BAL. Neither of
these pathologic repertoires overlapped with sequences present in blood
before patch test application. Similar to previous studies
(19), peripheral blood T cells contained no clonal
populations and no identifiable beryllium-reactive TCRs.
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). For example, one TCRBV14 sequence,
BV14-KGGG-BJ1S5, accounted for 2 of 27 (7.4%) sequences in the skin
and 3 of 37 (8.1%) sequences in the BAL. In addition, this sequence
was also present in the blood CD4+ T cell
population (1 of 35 sequences). The largest TCRBV14 clonal expansion in
BAL was also present in the skin biopsy. In patient 3, we also examined
the V3 subset to see whether the similarity in skin and BAL
repertoire after beryllium stimulation extended beyond the V regions
expanded in BAL. The percentage of
V3+CD4+ T cells in the
BAL and blood of patient 3 were not different (4.1 and 5.2%,
respectively) (Fig. 1). Similar to the findings for the expanded T cell
subsets, TCRBV3 sequence matches were present (Fig. 4). For example,
one TCRBV3 sequence, BV3-MRGGG-BJ2S2, accounted for 9 of 38 BV3
sequences (24%) in the lung and 5 of 26 (19%) in the skin. For this
subset, nearly 31% of the skin sequences were present in the BAL
repertoire and, conversely, 73% of the BAL sequences were present in
the skin. Mobilization of beryllium-reactive T cells into blood after patch testing
Previous studies have shown that beryllium-reactive T cells in the
BAL of patients with CBD are rarely present in the circulating pool
(18, 19). In the present work, we noted that this
compartmentalization was upset after patch testing. TCRBV14, BV3, and
BV2 sequences expressed by peripheral blood CD4+
T cells in patient 3 before and after BeSO4 patch
testing are shown in Fig. 5 (BAL
sequences are shown again for reference). Before patch testing, 1 of 35
BV14, 1 of 18 BV3, and 0 of 28 BV2 sequences in blood matched those
present in BAL. Remarkably, at day 35 following patch testing, 63% of
the blood TCRBV14 sequences were present in BAL and a similar
percentage of BAL sequences were present in blood (Fig. 5). The largest
BAL TCRBV14 clonal expansion, BV14-PKPTGVG-BJ2S6, was not detected in
the blood CD4+ population initially, but
following patch testing this sequence accounted for 22% of the TCRBV14
repertoire. For the BV3 subset, each of the three large clones in BAL
were detected in blood after patch testing, but the extent of overlap
between the blood and BAL repertoires was less than that seen for the
BV14 subset. For the BV2 subset, none of the expanded BAL clones was
detected in blood after patch testing.
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). The frequency of TCRs matching in
blood and BAL increased from 63 to 72% for BV14, from 9.4 to 53% for
BV3, and from undetectable to 46% for BV2. The in vitro expansion of
several BV3 and BV2 clones, initially identified in BAL, was
dramatic.
The presence of these new sequences in the circulation suggested that
functional responses of peripheral blood T cells to beryllium might be
altered after patch testing. To test this possibility, PBMCs from
patients 1 and 2 before and after patch testing were cultured in the
presence BeSO4 for 5 days (Fig. 6). Proliferative responses of cells from
patient 1 were not detectable (stimulation index <2) before patch
testing. These cells proliferated normally in response to PHA (data not
shown). In contrast, when blood was obtained from patient 1 on day 14
after patch testing, proliferation to each concentration of
BeSO4 was apparent with stimulation indices as
high as 14 at the previously determined optimal 1 x
10-5 M BeSO4
(19). The extent of proliferation of cells from patient 2
also appeared to be significantly enhanced when obtained after patch
testing, although the higher background proliferation resulted in very
little change in stimulation indices. Peripheral blood cells
from patient 3 obtained at day 35 following the
BeSO4 patch test proliferated vigorously in
response to 1 x 10-5 M
BeSO4 (peak thymidine incorporation, 63,261
± 2,156; stimulation index = 221) (data not shown in Fig. 6).
Unfortunately, blood from day 0 was not available for comparison.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Few lymphocytes are present in the skin of healthy individuals. Within 96 h of applying BeSO4 onto the skin of individuals sensitized to beryllium, inflammation accompanied by infiltration with CD4+ T cells occurs. Similar to previous studies (25), all individuals with CBD demonstrated erythema, induration, and a vesicular reaction typical of a contact dermatitis. This inflammation was beryllium specific in that it did not occur when saline was used instead of BeSO4, and, although not tested in this study, inflammation has not been seen after beryllium patch testing of control individuals who do not have a history of beryllium exposure or evidence of beryllium-reactive T cells (25). Although clonal T cell populations were present in skin early after patch testing, none of the TCRs expressed by these cells matched those present in BAL of the same patients or was recognizable as beryllium-specific when compared with past studies (19, 30). Still, it seems likely that a subset of these early infiltrating cells were beryllium specific. It is possible that the few Ag-specific cells were masked by a larger number of nonspecific T cells. In the setting of an inflammatory response, the production of chemotactic factors directs migration of activated effector CD4+ T cells to sites of inflammation, regardless of specificity (31, 32). As a related example, mice immunized with myelin basic protein demonstrated minimal enrichment of pathogenic T cell subsets in inflammatory brain lesions (33). Instead, T cells at the site of pathology expressed heterogeneous TCRs, with the pathologic T cell subset being obscured by the influx of nonspecific T cells (33). It is possible that culturing of the early skin T cells with IL-2 would have enriched for the subset of Ag-specific T cells as has been shown in studies of EAE (34) and sarcoidosis (35).
Our studies of early skin lesions also provide an important control for
T cells present later in the granulomatous process. Thus, the
subsequent appearance of repertoires enriched for BAL-like TCRs cannot
be attributed to nonspecific recruitment of previously activated cells
to sites of inflammation. Considering the enormous diversity of the T
cell repertoire, finding T cells expressing identical TCRs in different
target organs indicates selection by the same Ag, in this case
beryllium. We were also able to identify TCRs documented to be
beryllium specific based on previous studies of cloned
V3+ T cells from BAL. These TCRs are
characterized by a -chain CDR3 motif with an invariant aspartate (D)
at position 96 and a particular length (19, 30). Although
not analyzed in this study, the paired TCR -chain is also highly
related among the cells that express this beryllium specific -chain.
Although only one of the three patients in the current study had BAL T
cells with this TCR, previous studies have demonstrated similar (or
even identical) TCRs in almost all individuals with CBD caused by
exposure to beryllium in the ceramics industry (30).
Patients 2 and 3 in the current study were exposed in the nuclear
weapons industry.
The presence and expansion of CD4+ T cells in skin that express TCRs identical to those in BAL provides new insight regarding the pathogenic potential of these cells. Infiltration into tissue in association with the granulomatous process strongly suggests that these CD4+ T cells do the same in lung tissue. Our studies also show that these cells have the capability of recognizing beryllium in other tissues, and it seems likely that granulomas in liver and lymphatic tissue in CBD involve the same subset of cells. It is currently not clear whether the pathogenic T cells arrive early in the skin and then expand over the next 2–5 wk or whether their arrival and expansion in the tissue are continuous during the generation of granulomas. The latter is supported by the high frequency of these cells in the circulating pool later after patch testing.
The granulomatous inflammation in CBD is identical to that in
sarcoidosis, and these diseases share a number of clinical
characteristics (36). The development of granulomatous
inflammation in the skin following patch testing with
BeSO4 is similar to the Kveim-Siltzbach reaction
seen in patients with sarcoidosis (37, 38). Following the
intradermal injection of the Kveim-Siltzbach reagent (a suspension of
sarcoid tissue extract), noncaseating granulomatous inflammation
develops after 4 wk. Analysis of TCR -chain repertoire of these
reactions showed oligoclonal T cell populations consistent with a
conventional Ag response (38). Compared with BAL T cells
from sarcoidosis patients in general, similarities in the skin TCR CDR3
regions were noted to be suggestive, but analysis of BAL TCR repertoire
in the same individuals was not performed and identical TCRs were not
found. Our study is unique in this regard.
The majority of CBD patients will have blood cells that test positive
in a beryllium lymphocyte proliferation assay (1). Thus,
these individuals have beryllium-specific T cells in their circulating
pool. Still, we have noted that there is a separation of lung and
peripheral blood CD4+ TCR repertoires in these
patients (19). Precursors or progeny of clonally expanded
BAL T cells are absent or extremely rare in the circulating pool. The
same was true in the present study, when blood T cells were analyzed
before patch testing. These results likely relate to the low frequency
of blood T cells that proliferate in response to beryllium in culture,
which is below the frequency detectable by TCR sequence analysis.
However, it is also possible that blood and BAL beryllium-specific T
cells are separate populations with separate TCRs. In the present
study, after 2–5 wk following patch testing, we noted a marked
increase in T cells with TCRs identical to those expanded in BAL. The
beryllium-specific nature of these cells is further supported by the
enhanced proliferation of peripheral blood lymphocytes and the further
expansion of particular T cell clones after in vitro stimulation with
beryllium. Only considering the three TCRBV subsets (which account for
20% of the repertoire), our results suggest that at least 2% of
the peripheral blood CD4+ T cells may be
beryllium specific after patch testing. This frequency of Ag-specific
CD4+ T cells has only been reported for some
patients with chronic viral infections (39). In contrast,
memory CD4+ T cells specific for tetanus toxoid
or purified protein derivative of Mycobacterium tuberculosis
in healthy individuals occur at a frequency of 1 in 5,000 to 1 in
100,000 CD4 cells (40). Estimates of Th cell precursor
frequency in the blood following injection of a conventional Ag like
keyhole limpet hemocyanin ranged from 1 in 100,000 to 1 in 800,000
(41).
Our results show that application of BeSO4 to the skin of individuals with CBD results in a mobilization of beryllium-specific CD4+ T cells into the circulating pool. Depending on the degree of systemic Ag exposure after patch testing, the lymphoid tissue draining the patch test site or even the peri-lung lymphoid tissue may be the sites contributing to the circulating cells. The results prompted us to reevaluate the safety of patch testing in CBD patients. The safety of patch testing has been previously questioned (20, 21). More recent studies have found no evidence of clinical deterioration or progression related to patch testing (25). Additional follow-up of the same patients indicated no evidence of disease exacerbation (L. S. Newman, unpublished observation). Furthermore, no clinical changes have been noted in the three individuals patch tested in the current study, and follow-up is being continued. It should be noted that we deliberately selected study subjects for patch testing who no longer worked in the beryllium industry, as a further safeguard against the theoretical possibility of exacerbating the beryllium-induced immune response. However, the increased number of circulating beryllium-specific T cells after patch testing suggests the possibility that disease exacerbation may occur after patch testing, although none has been seen in the small number of patients followed to date.
Together, our results indicate that patch testing provides an in vivo model of granulomatous disease in individuals with CBD. We were able to observe the progression from influx of nonspecific T cells to the presence and expansion of beryllium-specific CD4+ T cells at the time of granuloma formation. The kinetics of this process cannot be studied in the organs of individuals with established disease and in vitro studies cannot provide the same insight shown in this work. Whether patch testing can be used in non-granulomatous diseases thought to be mediated by CD4+ T cells remains unknown. For example, patch testing may be useful to mobilize and characterize pathogenic CD4+ T cells in diseases where these cells are sequestered in target organs, such as the brain in multiple sclerosis, pancreas in insulin-dependent diabetes mellitus, or even joints in rheumatoid arthritis. Obviously, the safety of this procedure in these diseases would need to be addressed in a rigorous fashion.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew P. Fontenot, Department of Medicine, Division of Clinical Immunology (B164), University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: andrew.fontenot{at}uchsc.edu
3 Abbreviations used in this paper: CBD, chronic beryllium disease; BeSO4, beryllium sulfate; BAL, bronchoalveolar lavage; CDR, complementarity-determining region; TCRB, TCR -chain gene.
Received for publication September 6, 2001. Accepted for publication January 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene usage in the central nervous system in experimental allergic encephalomyelitis. J. Immunol. 150:4085.[Abstract]
gene usage in myelin basic protein reactive T cell clones from patients with multiple sclerosis. Proc. Natl. Acad. Sci. USA 88:9161.-specific T cells in the intradermal response to Kveim-Siltzbach reagent in individuals with sarcoidosis. J. Immunol. 154:1450.[Abstract]
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