| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Departments of Human Developmental Biology and Immunology, Jagiellonian University College of Medicine, Krakow, Poland;
Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; and
Noda Institute for Scientific Research, Noda-shi, Chiba-ken, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(2), which induce local tissue cells to produce chemokines that recruit and activate an infiltrate of bone marrow-derived leukocytes (1).
Local endothelial cell activation plays a crucial role. Vasopermeability is induced (3) and can be evaluated by measuring ear swelling 2 h after Ag challenge (4). Also, T cell recruitment is crucially aided by increased expression of endothelial adhesion molecules like ICAM-1 and VCAM-1 (5), which is due to mast cell-derived TNF-
(5, 6). The exact cascade of events responsible for mast cell-dependent endothelial activation has been a puzzle because the mere presence of Ag, or even local Ag challenge plus locally circulating immune T cells, does not account for the T cell recruitment (7, 8, 9). Therefore, we have focused on this early DTH-initiating phase to determine the processes needed for endothelial activation required for T cell recruitment.
In recent studies of initiation of CS responses, we noted activation of complement C5 (10, 11) to generate C5a (6) to stimulate local mast cells (3, 4, 12, 13, 14) and likely platelets (15). Elaborated C5a likely activates mast cells via their C5a receptors (6, 16) to release vasoactive serotonin (8, 12, 14) and TNF- (5, 6), which are required for endothelial activation and subsequent extravasation of the circulating T cells (3, 6, 8, 9, 11, 12). Furthermore, recent findings show that B cells also are involved in the initiation process in CS (9, 11), which was thought previously to be solely caused by T cells (1). These CS-initiating B cells are B-1 cells that are activated within 1 day after immunization to produce IgM Abs, which early in the elicitation phase bind local challenge Ag, leading to complement activation to generate C5a (6), which activates mast cells for T cell recruitment.
CS and DTH differ in several important aspects. Because CS initiation was shown in models involving contact skin immunization with haptens, like picryl chloride (6, 9, 10, 11), oxazolone (7), and 2,4-dinitro-1-fluorobenzene (4, 17) or dinitro-chlorobenzene (13, 18), and also with reactive simple metal contact sensitizers like nickel and chromium (18), we determined whether similar initiating processes occur in DTH to soluble protein Ags (1). This was judged to be important because DTH to soluble protein Ag is thought to participate in clinically important forms of T cell-mediated immunity such as allergies, autoimmunity, and microbial and tumor resistance. We used a recently established protein DTH system in which mice are immunized intradermally (i.d.) with protein Ags in saline (39). This approach avoided adjuvants, which stimulate other overlapping responses not essential to DTH that might complicate interpretation of results. Furthermore, the use of adjuvants was considered less natural compared with exposure to Ag in clinical allergies, autoimmunity, and tumor immunity. Also, we studied DTH elicited only 4 days after immunization, (39) before T cell-dependent B-2 cell Ab responses might arise (1), which could overlap DTH and confuse findings. DTH elicited on day 4 is Ag specific and caused by 
T cells with a cytokine profile suggesting that Th1 and Th2 or Th0 cells contribute (39).
In the current study, we show that elicited DTH indeed involves an early DTH-initiating cascade, like in CS. Similarly, IgM, complement, C5a, and mast cells were found to participate in recruitment of effector T cells. Importantly, we found involvement of B-1 B cells rapidly producing IgM as early as 1 day after immunization.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Six- to eight-week-old specific pathogen-free male CBA/J; female BALBc/J, immunodeficient TCR-/- (C57BL/6 background), virtually pan B cell-deficient (19) µMT (C57BL/6), C5aR-/- (20), and IgM-/- (both B6 129SF2/J background) (21), male CBA CaHN-btk-xid/J (CBA/N-xid or xid) mice deficient in B-1 cells (22), and mast cell-deficient WBB6F1/J-KitW/KitW-v and control wild-type mice (C57BL/6) were obtained from The Jackson Laboratory (Bar Harbor, ME). Some experiments were performed in CBA/J bred at the Jagiellonian University College of Medicine (Krakow, Poland). Mating pairs of pan B cell-deficient JH-/- mice (CB.17/BALB/c background) (23) were from Mark Shlomchik (Yale School of Medicine, New Haven, CT). Immunodeficient mice were fed autoclaved food and water under pathogen-free conditions using filter-topped microisolator cages and sterile equipment. Mice were rested for 1–2 wk before use. Experiments were conducted according to guidelines of the Animal Care and Use Committee.
Reagents
Keyhole limpet hemocyanin (KLH) was obtained from Sigma-Aldrich (St. Louis, MO) and Calbiochem (San Diego, CA). OVA was from Sigma-Aldrich. The concentration of endotoxin in KLH preparations was measured by Limulus Amebocyte Lysate assay (Associates of Cape Cod, Falmouth, MA). Sigma-Aldrich KLH contained 2420 endotoxin units (EU)/mg protein, and Calbiochem KLH had substantially less endotoxin (1–19 EU/mg protein). Within these limits of ±10–2420 EU/mg, the amount of LPS in KLH did not influence DTH responses (39). Also, equivalent responses were obtained in LPS-responsive C3H/HeN and in LPS-resistant C3H/HeJ male mice (The Jackson Laboratory) (39).
Monoclonal Abs and complement
Monoclonal Abs from BD PharMingen (San Diego, CA) were: anti-CD4 (clone GK1.5), CD8a (53-6.7), TCR (H57-597), TCR
(UC7-13D5), Thy1.2 (53-2.1), CD5 (53-7.3), CD19 (1D3), CD22 (Cy34.1), Mac-1 (M1/70), and CD45RA/B220 (RA3-6B2), which were used at optimal concentrations recommended by the manufacturer. Low-tox rabbit C was from Pel-Freez Biologicals (Brown Deer, WI).
Active immunization and skin testing to elicit DTH
Mice were immunized by i.d. injection of 100 µg of KLH or OVA diluted in 200 µl of freshly opened sterile pyrogen-free 0.9% saline (Abbott Laboratories, North Chicago, IL), injected without added adjuvants into four divided sites in the alcohol-cleaned and unshaved abdominal skin (39). For low-responder C57BL/6 background mice, we used i.d. 100-µg immunizations on two consecutive days. Littermate controls were injected identically with the saline diluent. On day 4, ear swelling was elicited under ether anesthesia via i.d. injection in both ears with 10 µl containing 5 µg of KLH or OVA in saline. Controls were "immunized" with saline alone and ear challenged with KLH or OVA. Resulting thickness of the Ag-challenged ears was measured with a micrometer (Mitutoyo, Kanagawa, Japan) before and at 2 and 24 h after challenge. Increased ear thickness was expressed as mean (in millimeters) x 10-2 ± SE (19). Some responses are expressed as net increased ear thickness by subtracting KLH ear-challenge responses of controls immunized with saline from those of KLH-immunized and KLH-ear-challenged experimental mice.
Adoptive cell transfer of 2- and 24-h components of DTH
CBA/J immune cell donors were sensitized i.d. with KLH. On day 1 or 4, lymph nodes and spleens were harvested and single cell suspensions were prepared. Then a mixture of 7 x 107 immune lymph node and spleen cells in 0.25 ml of PBS were adoptively transferred i.v. into syngeneic naive recipients that were ear challenged with KLH 1 day later. In cell depletion experiments, 4-day immune cells were incubated with mAb for 45 min on ice, washed, and then incubated in diluted rabbit C at 37°C for 1 h. After incubation and washing, 7 x 107 mixed immune lymph node and spleen cells treated with C alone, and cell equivalents remaining after treatment with different mAb plus C, were adoptively transferred i.v. into naive recipients. Then 24 h later, recipients were ear challenged with KLH and tested for 2- and 24-h DTH.
Isolation of peritoneal exudate cells (PECs)
In some experiments, PECs were used as a source of KLH immune cells for transfer. PECs were induced by i.p. injection of 2 ml of thioglycolate medium (Difco, Detroit, MI) on the day of immunization, collected 4 days later by washing the peritoneal cavity with ice-cold PBS containing 5 U of heparin/ml, and then washed with PBS. Then the whole population, or the nonadherent fraction, was transferred i.p. into naive recipients that were tested for DTH 1 day later. To isolate the nonadherent population, 5 x 107 PECs in 8 ml of RPMI 1640 medium with 5% FCS were adhered to 100-mm plastic petri dishes (Fisher Scientific, Pittsburgh, PA) for 2 h at 37°C and 5% CO2. Dishes then were gently rotated and nonadherent cells were harvested.
FACS sorting of immune lymphoid B-1 vs B-2 cells
One-day KLH immune BALB/c mixed spleen and lymph node cells at 107/ml were stained with anti-CD5-cychrome and anti-CD19-FITC at 0.025 µg per 106 cells (BD PharMingen) for 30 min on ice and then washed with 4°C RPMI 1640 without FCS. Stained cells were sorted with a FACSVantage SE (BD Biosciences, San Jose, CA) to obtain CD19+CD5+ B-1 and CD19+CD5- B-2 cells (each ±98% enriched). Then, 2.5 x 104 B-1 cells or 6 x 106 B-2 sorted cells were transferred i.p. into 3-day KLH immune pan B cell-deficient JH-/- recipients, and 1 day later they were ear challenged with KLH and tested for 2- and 24-h DTH.
Transfer of the 2-h component and consequent DTH initiation with 1-day immune serum
CBA/J donors were immunized i.d. with KLH and the next day were bled. Collected blood was kept for 30 min at 25°C and then placed at 4°C. The next day, serum was collected from retracted clotted blood and frozen at -20°C until used. Subsequently, 3-day KLH immune B-1 cell-deficient CBA/N-xid mice were injected i.v. with graded volumes of the 1-day KLH immune serum or with control serum from naive donors. The next day, recipients were ear challenged with KLH and tested for 2- and 24-h DTH.
Monoclonal inhibition of C5 in vivo
Four-day KLH immune CBA/J mice were injected i.v. with saline alone (positive controls) or with rat anti-mouse C5 IgG1 mAb (BB5.1), or isotype control Ab (135.8 anti-human C8 IgG1; Alexion, Cheshire, CT) 1 mg/mouse 1 h before or 3 h after KLH ear challenge. Then mice were tested for DTH.
ELISPOT assay for anti-KLH IgM-producing cells
Spleen cells from 1-day and 4-day KLH immune mice were seeded in triplicate into 96-well filtration plates with Immuobilon-P membranes (Millipore, Bedford, MA) at 2 x 106 cells/well and precoated with 50 µl of KLH (50 µg/ml). Plates were incubated overnight at 37°C, cells were discarded, and wells were washed with PBS three times and with PBS containing 0.05% Tween 20 three times and then were incubated for 1 h at 25°C with 2 µg/ml biotin-conjugated anti-mouse IgM mAb (BD PharMingen) followed by incubation with streptavidin-HRP for 1 h (1/200; Vector Laboratories, Burlingame, CA). Spots were developed using 3-amino-9-ethylcarbazole as substrate, the reaction was stopped by washing, and wells were dried at 25°C in the dark. Membranes were removed and stuck on glass slides, and spots were enumerated under an inverted dissecting phase microscope and expressed per organ.
Statistics
The paired two-tailed Student t test was used with p < 0.05 taken as the level of significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Four days after i.d. immunization with 100 µg of KLH in saline, 24-h DTH was elicited by i.d. injection of 5 µg of KLH in saline into the ear skin bilaterally, but not in KLH ear-challenged controls sham immunized with saline (Fig. 1a, Groups A and B, right). Also, at 2 h after local challenge, there was significant ear swelling in KLH-immunized and KLH-ear-tested mice, compared with challenged saline-immunized mice (Fig. 1a, Groups A and B, left).
|
Furthermore, the 2-h component of DTH also was Ag-specific, because the KLH-induced 2-h early component was elicited by KLH but not OVA challenge (Fig. 1b, Groups B and C, left). Moreover, OVA but not KLH challenge elicited significant 2-h ear swelling in OVA-immunized mice compared with saline-immunized mice (Fig. 1b, Groups D and E, left) and compared with OVA-immunized and KLH-challenged mice (Groups E and F, left). We concluded that DTH induced by i.d. KLH or OVA protein injection was Ag specific in both the classical 24-h T cell component and the 2-h response.
Adoptive cell transfer of 2- and 24-h components and the phenotype of cells mediating both aspects of DTH
Injection i.v. of lymph node or spleen cells from immunized CBA/J (H-2k) donors adoptively transfers 24-h DTH to nonimmune mice (Fig. 2a, Groups A, D, and E, right). Also, whole 4-day PECs adoptively transferred 24-h ear swelling (Fig. 2a, Group B, right), as did the nonadherent fraction with macrophages removed (Fig. 2a, Group C, right). In addition, these 4-day immune populations also transferred the ability to elicit the 2-h DTH component. Indeed, immune lymph node or spleen cells, immune whole PECs, or the PEC nonadherent fraction all adoptively transferred significant 2-h reactivity (Fig. 2a, Groups B–E, left).
|
T cells by treatment in vitro with anti-TCR mAb plus C did not transfer 24-h responses, but still were able to transfer 2-h reactivity (Fig. 2b, Group C), suggesting that the 2-h component was not caused by T cells. Furthermore, CD4-depleted cells failed to transfer 24-h DTH reactivity (Fig. 2b, Group D, right), but again 2-h activity was unaffected (Fig. 2b, Group D, left), whereas anti-CD8 plus C depletion had no effect on either 2- or 24-h transfers (Fig. 2b, Group E). It was concluded that CD4+CD8- TCR-+ T cells were largely responsible for the transfer of 24-h DTH reactivity. In contrast, the cells responsible for 2-h reactivity were CD4-, CD8-, and TCR--, and thus were not T cells. Functional phenotype of immune lymphoid cells mediating 2-h DTH
To further determine the phenotype of cells transferring the 2-h component, 4-day immune cells were treated in vitro with additional mAbs and C, washed, and adoptively transferred i.v., and recipients were ear challenged and tested for DTH. The mAbs chosen were mainly against determinants on B cells because the early component cells were not T cells (Fig. 2b).
Fig. 3 shows that the 2-h component of DTH may be caused by B-1 cells, because the mediating cells are CD5+, CD19+, CD22+, and B220+ (Groups A, C–E, and G, left), which also express Mac-1 and Thy-1.2 (Groups B, left, and F), but again not CD8 (Group H). These determinants were found previously on B-1 cells that mediate the 2-h initiating component of CS (17, 18, 24). However, further study was needed because this analysis could not conclude whether determinants detected were on the same cell or on different collaborating cells.
|
T cells. The 2-h component of DTH is absent in B-1 cell-deficient xid mice and depends on mast cells
We attempted to confirm that B cells are important in DTH initiation. Fig. 4 shows genetic evidence that B cells are the DTH-initiating cells. Active KLH immunization in µMT virtually B cell-deficient mice did not elicit 2-h DTH responses, and probably as a consequence, no 24-h DTH could be elicited (Fig. 4, Groups A and C). This formulation was confirmed when we examined the potential role of B-1 cells by immunizing predominately B-1 cell-deficient CBA/N-xid male mice vs intact CBA/J male mice. CBA/J mice elicited strong 2- and 24-h DTH, whereas B-1 cell-deficient xid mice elicited neither 2- nor 24-h DTH responses (Fig. 4, Groups F and G), implicating B-1 cells in mediation of DTH initiation. Furthermore, we compared responses in TCR-/- mice to those in background B6 controls. As expected, 24-h DTH was not elicited in TCR-/- mice (Fig. 4, Groups A and B, right) because of a lack of DTH-effector T cells. Remarkably, there was nearly full elicitation of the early 2-h component of KLH-induced DTH in TCR-/- mice (Fig. 4, Groups A and B, left).
|
T cells to elicit subsequent classical 24-h late DTH. The early 2-h DTH component can be transferred with 1-day KLH immune cells
The initiation phase of CS is caused by B-1 cells induced only 1 day postimmunization (4, 7, 9, 25, 26). Thus, we determined the time post-KLH i.d. immunization needed to induce 2- vs 24-h DTH. We attempted to elicit responses in 1-day vs 4-day actively immune mice and also in naive recipients after adoptive transfer of 1-day vs 4-day KLH immune cells. As before, mice elicited both 2- and 24-h components of DTH 4 days after active KLH i.d. immunization (Fig. 5a, Group A), and adoptive transfer of 4-day immune cells resulted in ability to elicit both 2- and 24-h DTH components (Fig. 5a, Group B). Importantly, by only 1 day postimmunization, actively KLH-sensitized mice manifested the 2-h early component, but without the 24-h T cell late response (Fig. 5a, Group C), and cells harvested from similar 1-day immune mice, when transferred i.v. to naive recipients, also elicited the 2-h component, again without the 24-h response (Fig. 5a, Group D).
|
T cells that have not yet developed (Fig. 5a, Groups C and D). One-day KLH immune cells reconstitute DTH initiation in B cell-depleted 4-day immune cells
In a cell transfer system, we determined whether 1-day KLH immune cells could mediate initiation by reconstituting 24-h DTH when transferred with 4-day immune cells depleted of B cells. Untreated positive control 4-day KLH immune CBA/J cells transferred both 2- and 24-h DTH (Fig. 5b, Group A). In contrast, depletion of B cells by treatment in vitro with anti-CD19 plus complement before transfer eliminated transfer of both 2- and 24-h DTH (Fig. 5b, Group B). Again, 1-day KLH immune cells transferred the 2-h but not the 24-h component (Fig. 5b, Group C). Finally, a combined transfer of 4-day immune T cells depleted of B cells, together with 1-day KLH immune cells, caused elicitation of the 2-h component, allowing elicitation of the classical 24-h T cell component of DTH, due to the B cell-depleted 4-day immune T cells (Fig. 5b, Group D).
FACS-sorted 1-day KLH immune B-1 cells reconstitute elicitation of 24-h DTH in KLH-immunized JH-/- recipients
We attempted to unambiguously demonstrate that B-1 cells were responsible for early 2-h initiation of 24-h late DTH-effector T cells. Recipients were pan B cell-deficient JH-/- CB.17 H-2d mice, which were actively KLH-sensitized to attempt induction of KLH-specific effector T cells in the absence of all B lymphocytes. To these 3-day KLH-immune JH-/- recipients we adoptively transferred 1-day KLH immune cells from nearly syngeneic BALB/c H-2d donors. In other groups, we also transferred FACS-sorted B-1 (CD19+, CD5+) vs B-2 cells (CD19+, CD5-) purified from the 1-day immune cells. As before, 1-day KLH immune cells transferred 2-h but no 24-h DTH activity (Fig. 5c, Group B), compared with JH-/- control nonimmune challenge recipients (Group A). This ruled out that the 1-day immune cells directly mediated the 24-h responses. Furthermore, 1-day KLH immune BALB/c cells transferred 2-h ear swelling activity that allowed 24-h late responses in the previously KLH-immunized JH-/- recipients, likely mediated by immune JH-/- effector T cells (Fig. 5c, Group C). Importantly, sorted B-1 cells in the 1-day immune cells similarly mediated 2-h responses, which also reconstituted elicitation of 24-h late DTH (Fig. 5c, Group D), whereas the B-2 cell fraction did not (Fig. 5c, Group E). We concluded that the 2-h component was caused by Ag-specific B-1 cells, which became activated within 1 day of immunization and were required to mediate recruitment of effector T cells to elicit classical 24-h DTH.
One-day KLH immune serum reconstitutes the 2-h component, allowing 24-h DTH
We tested whether serum Abs produced by B-1 B cells are able to initiate KLH-induced DTH. CBA/N-xid H-2k B-1 cell-deficient mice were immunized i.d. with KLH on day 0. Three days later, they received i.v. transfer of 1-day KLH immune cells, or were injected i.v. with serum from naive CBA/J mice as a control, or received graded volumes of serum from CBA/J mice that were immunized with KLH 1 day before bleeding. Immunized B-1 cell-deficient xid mice developed neither the 2-h nor the full 24-h component of DTH (Fig. 6, Group A). In contrast, both the 2- and 24-h responses were reconstituted fully by transfer of 1-day immune cells from wild-type mice (Fig. 6, Group A vs B) or by 1-day immune serum at doses from 0.75 ml to 0.25 ml per recipient, but not by 0.075 ml of immune serum (Fig. 6, Groups D–F). In contrast, injection of 0.75 ml of normal serum did not reconstitute either early or late components of DTH (Group G vs A). Finally, injection of 1-day immune serum into naive unimmunized xid mice just reconstituted the 2-h component (Group C). These results show that serum Ab likely produced by B-1 cells by 1-day postimmunization is required to initiate KLH-induced DTH and thus to recruit sensitized 24-h effector T cells induced in immunized xid mice.
|
The data in µMT and B-1-deficient xid mice and the ability of 1-day KLH immune serum to reconstitute DTH initiation in B-1-deficient mice suggest that B-1 cell-derived IgM Ab is involved. To determine whether IgM participates in DTH initiation, we used mice solely unable to secrete IgM (IgM-/- mice, 129 background) (21). KLH-immunized IgM-/- mice failed to elicit both 2-h and 24-h components of DTH (Fig. 7a, Groups B vs F), showing that IgM likely is crucial in DTH initiation.
|
To confirm involvement of C5 and to determine whether C5 acts in the early DTH initiation process, we attempted to neutralize C5 in vivo in wild-type mice. Accordingly, mice treated with anti-mouse C5 mAb 1 h before KLH ear challenge (Fig. 7b, Group C), but not with saline (Group B) or isotype control Ab (Group D), had significantly decreased 2- and 24-h DTH. In contrast, anti-C5 treatment performed 3 h after KLH ear challenge, and thus after the 2-h initiating component, had no effect on elicited DTH (Fig. 7b, Group F).
Early specific IgM Ab responses are impaired in xid mice
We used ELISPOT to evaluate numbers of spleen cells producing anti-KLH IgM Abs after KLH immunization in wild-type compared with xid mice. There were background responses in sham/saline immune CBA/J mice (Fig. 8, Group A), but not in sham xid mice (Group D). Furthermore, we found significantly increased numbers of anti-KLH IgM-producing cells at 1 day and 4 days after KLH skin immunization in CBA/J mice (Fig. 8, Groups B and C), compared with xid mice (Groups E and F). Our findings of impaired production of specific IgM Abs at days 1 and 4 in xid mice reinforced the idea that early activated B-1 cells produce DTH-initiating IgM Abs.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T cell-mediated 24-h DTH to a soluble protein Ag is preceded by an early 2-h component, 2) the early component is caused by B-1 B cells that become activated rapidly by just 1 day after immunization, and 3) the B-1 cell-mediated early DTH process involves rapid production of specific serum IgM Ab, mediating complement activation early post-Ag challenge. This generates C5a to stimulate local mast cells via C5a receptors and is DTH initiating, because it is required to recruit effector T cells to mediate classical 24-h DTH. A very similar process previously was noted as required to initiate 24-h CS, also via T cell recruitment (6, 7, 8, 9, 10, 11). DTH and CS are important in vivo manifestations of T cell immunity. Elicitation of both responses depends on several cell types that contribute to the final local inflammatory and tissue swelling response. The current study examined whether there is an early initiating phase in soluble protein Ag-induced DTH necessary to recruit DTH-effector T cells. First we showed, similar to CS, that mice actively immunized and subsequently challenged i.d. with protein Ag on day 4 elicit both 2- and 24-h components of DTH. Additionally, 2- and 24-h DTH were Ag-specific because mice actively immunized with KLH or OVA, another soluble heterologous protein Ag, elicited both early and late DTH only when challenged with the Ag used for immunization.
Protein Ag-induced DTH could be transferred to naive syngeneic recipients with cells from actively immunized mice. Thus, by day 4 after i.d. immunization with soluble protein Ag in saline without adjuvants, sensitized cells develop that can mediate DTH, as shown previously in CS (4, 7). The current results show in addition that immunization with protein Ag also induces immune cells that mediate a required early DTH-initiating component. Experiments to characterize the cells mediating the 2-h vs 24-h component of DTH showed that the late component is mediated by CD4+CD8- TCR-+ effector T cells, whereas the early-acting 2-h DTH-initiating cells are Ag-specific triple negative CD4-CD8-TCR-- cells and thus are not T cells (24). Therefore, CD4+ and not CD8+ T cells mediate this DTH response that is induced by KLH i.d. sensitization and is elicited early postimmunization. Remarkably, these early-acting DTH-effector T cells produce both Th1-type and Th2-type effects that recruit a dominant eosinophil infiltrate (39).
To further characterize the phenotype of the cells mediating the early-acting DTH component, aliquots of 4-day KLH immune cells containing the 2- and 24-h cells were treated in vitro before transfer with mAbs previously found to deplete CS-initiating cells (24). These mAbs virtually eliminated both the 2-h and subsequent late 24-h component of DTH. These data not only further characterized the phenotype of early component cells such as CD5+, CD19+, CD22+, B220+, Mac1+, and Thy1+, but they also demonstrated the requirement for the early cell activity to elicit the late DTH-effector component mediated by T cells (Fig. 3).
Prior results in CS suggesting that the CS-initiating component is mediated by B-1 cells were paralleled in further new results in DTH. We studied KLH DTH induced in two B cell-deficient genetically modified strains, µMT and JH-/-, and in CBA/N-xid mice that principally lack the B-1 cell subset of B cells (22). Results showed defective elicitation of both early and subsequent late components of DTH in all three types of B cell-deficient mice, confirming the data obtained by mAb plus complement depletion. Moreover, defective elicitation of 2- and 24-h DTH responses in xid mice confirmed findings in CS (9) showing that initiation in protein Ag-induced DTH is likely mediated by B-1 cells. The fact that µMT mice on a B6 background have small IgA responses (19) should not influence our results.
Initiation of CS responses is mediated by specific IgM Abs rapidly produced by B-1 cells by 1 day postimmunization (9), which bind challenge Ag, activate complement, and release C5a to activate mast cell C5aR (6, 16) and platelets (15, 27) to release serotonin (8, 28, 29) and TNF- (5, 6, 30), allowing recruitment of effector T cells. To determine whether a similar initiating process occurs in DTH, we tested mast cell-deficient mice, which had a strongly reduced early 2-h component and total lack of late 24-h DTH, suggesting that mast cells play a role in elicited KLH DTH, as noted in several prior studies of CS (3, 13, 14, 31, 32, 33, 34).
Another issue pertinent to DTH initiation needed for T cell recruitment is the time required for induction of initiating B-1 cells, which in CS are rapidly induced within 1 day of immunization. Thus, we compared 1-day vs 4-day KLH actively immunized mice and measured both 2- and 24-h DTH. Mice tested 4 days after active immunization developed both early and late components, whereas mice challenged just 1 day after immunization developed only the 2-h response. Moreover, the 2-h component of DTH was adoptively transferred with 1-day KLH immune cells, suggesting that the early DTH-initiating B-1 cells are induced within 1 day in the protein Ag system, as in CS (4, 7, 9). Finally, we tested the actual DTH-initiating capacity of 1-day KLH immune cells in reconstitution of 24-h DTH mediated by 4-day KLH immune T cells depleted of CD19+ B cells. Restoration of both 2- and 24-h DTH confirmed that DTH-initiating cells are induced within 1 day and express CD19, a characteristic marker of all B cells. The experiments in virtually B cell-deficient µMT mice and in B-1 cell-deficient xid mice and negative selection with mAb plus complement of immune cells expressing markers of activated B-1 cells (CD5, CD19, B220 Mac-1, or Thy-1) suggest that B-1 B cells are critical to initiation of DTH.
To extend our findings, we attempted transfer of 2-h DTH-initiating activity into 4-day immune pan B cell-deficient JH-/- mice that could not elicit 24-h DTH. These mice generated DTH-effector T cells, but had no means of their local recruitment for 24-h DTH (9). We transferred either FACS-sorted 1-day immune B-1 (CD5+CD19+) or B-2 (CD5-CD19+) cells into the 4-day KLH-immunized JH-/- recipients. The result was that only CD5+CD19+ B-1 cells were able to transfer early 2-h DTH initiation, which allowed recruitment of T effector cells responsible for late component of DTH. This resulted in reconstitution of elicitation of 24-h responses likely mediated by locally recruited DTH-effector T cells induced by immunization of JH-/- mice, which could not be recruited because of the absence of DTH-initiating B-1 cells.
We suggested that activated B-1 cells produce the 2-h component of DTH via secreted IgM Ab, as in CS (9). This was confirmed in DTH by experiments showing that 1-day KLH immune serum transferred into naive CBA/N xid mice led only to the early component. In a further experiment, we showed that IgM-/- mice, which could not make secreted IgM responses (21) but were able to produce all other Ig isotypes, do not develop either the early 2-h or the 24-h late component of DTH. This further suggests that immunization-induced IgM Abs indeed play a crucial role in DTH initiation. These induced Ag-specific IgM Abs appear to be beyond the natural background IgM Abs in normal nonimmune sera (35), which include those specific for contact-sensitizing haptens (36). ELISPOT assay confirmed that KLH immunization for DTH also generated specific IgM Abs by only 1 day and also at 4 days. The absence of these anti-KLH IgM Ab responses in xid mice indicates that B-1 cells were involved.
Furthermore, in CS hapten-specific IgM locally activates complement to generate C5a to mediate CS initiation. To confirm whether a similar mechanism occurs in DTH, we tested C5a receptor-deficient (C5aR-/-) vs control mice. We found that C5aR-/- mice develop diminished early and late components of KLH DTH compared with controls. The role of complement C5 was confirmed in another independent experiment, showing that neutralization of C5 by systemic treatment with specific mAb significantly decreased the early 2-h and late 24-h components of DTH. Furthermore, the locus of C5 acting early in elicitation was confirmed by showing that anti-C5 was inhibitory when given before testing, but that it was not inhibitory when given just 3 h postchallenge when the DTH-initiating process had ended. A role for complement C5 recently has been shown in some experimental models of human diseases, in which mast cell activation for elicited DTH-like mechanisms are thought to participate, such as in the collagen arthritis model of rheumatoid arthritis (37, 38).
We concluded that protein Ag-induced DTH is very similar to CS in requiring an early initiation response that leads to local T cell recruitment (process summarized in Fig. 9). As in CS, initiation of DTH is mediated by Ag-specific B-1 cells that are activated just 1 day after i.d. immunization with KLH, which releases Ag-specific IgM Abs into the circulation. Subsequently, the IgM Abs present at the peripheral Ag challenge sites likely bind to the local challenge Ag to generate immune complexes that activate complement. This leads to local release of C5a required to activate C5aR to trigger mast cell- and platelet-mediated DTH-initiating vasoactive functions that are required for recruiting the DTH-effector T cells into the tissues to mediate the classical 24-h responses (Fig. 9). Finding DTH initiation that is similar to CS initiation raises the possibility that other DTH-related responses and other types of in vivo T cell-mediated effector responses, such as resistance to microbes or tumors as well as autoimmunity and tissue allergic responses, could similarly depend on early-occurring DTH-initiating events.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Philip W. Askenase, Section of Allergy and Clinical Immunology, Department of Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208013, New Haven, CT 06520-8013. E-mail address: philip.askenase{at}yale.edu
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; CS, contact sensitivity; i.d., intradermal; KLH, keyhole limpet hemocyanin; EU, endotoxin unit; PEC, peritoneal exudate cell.
Received for publication January 21, 2003. Accepted for publication September 22, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in delayed-type hypersensitivity mediated by Th1 clones. J. Immunol. 143:2887.[Abstract]
. J. Immunol. 162:1648.: a possible initiating role of B cells. J. Exp. Med. 186:1015.IIR+). Invest. Dermatol. 102:321.[Medline]
chain expression in developing B cells. Nat. Immun. 2:625.
, and CXCR3 chemokines. Immunology. In press.This article has been cited by other articles:
|
|
 |
|
  S. M. Kerfoot, M. Szczepanik, J. W. Tung, and P. W. Askenase Identification of Initiator B Cells, a Novel Subset of Activation-Induced Deaminase-Dependent B-1-Like Cells That Mediate Initiation of Contact Sensitivity J. Immunol., August 1, 2008; 181(3): 1717 - 1727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. U. Norman, S. Hulliger, P. Colarusso, and P. Kubes Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement J. Immunol., January 1, 2008; 180(1): 510 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Watanabe, M. Fujimoto, N. Ishiura, Y. Kuwano, H. Nakashima, N. Yazawa, H. Okochi, S. Sato, T. F. Tedder, and K. Tamaki CD19 Expression in B Cells Is Important for Suppression of Contact Hypersensitivity Am. J. Pathol., August 1, 2007; 171(2): 560 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Savitsky and K. Calame B-1 B lymphocytes require Blimp-1 for immunoglobulin secretion J. Exp. Med., October 2, 2006; 203(10): 2305 - 2314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Campos, M. Szczepanik, M. Lisbonne, A. Itakura, M. Leite-de-Moraes, and P. W. Askenase Invariant NKT Cells Rapidly Activated via Immunization with Diverse Contact Antigens Collaborate In Vitro with B-1 Cells to Initiate Contact Sensitivity J. Immunol., September 15, 2006; 177(6): 3686 - 3694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Itakura, M. Szczepanik, R. A. Campos, V. Paliwal, M. Majewska, H. Matsuda, K. Takatsu, and P. W. Askenase An Hour after Immunization Peritoneal B-1 Cells Are Activated to Migrate to Lymphoid Organs Where within 1 Day They Produce IgM Antibodies That Initiate Elicitation of Contact Sensitivity J. Immunol., December 1, 2005; 175(11): 7170 - 7178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. W. Askenase, A. Itakura, M. C. Leite-de-Moraes, M. Lisbonne, S. Roongapinun, D. R. Goldstein, and M. Szczepanik TLR-Dependent IL-4 Production by Invariant V{alpha}14+J{alpha}18+ NKT Cells to Initiate Contact Sensitivity In Vivo J. Immunol., November 15, 2005; 175(10): 6390 - 6401. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
