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The route of antigen entry determines the requirement for L-selectin during immune responses.

Catalina MD, Carroll MC, Arizpe H, Takashima A, Estess P, Siegelman MH - J. Exp. Med. (1996)

Bottom Line: Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice.In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes.These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235-9072, USA.

ABSTRACT
L-selectin, an adhesion molecule constitutively expressed on leukocytes, is important for primary adhesion and extravasation of lymphocytes at specialized high endothelial venules within lymph nodes and other leukocytes at sites of inflammation. We have generated L-selectin-deficient mice by targeted disruption, and have confirmed a previously reported phenotype which includes strikingly impaired contact hypersensitivity (CHS) responses to reactive haptens (Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. J. Exp. Med. 181:2259-2264; Xu, J.C., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. 183:589-598.). Since the mechanism of this impairment has not been clarified, we sought to define the stage(s) at which the CHS response is affected in L-selectin-deficient mice. We show that epidermal Langerhans cells in L-selectin-deficient mice are normal in number, migrate to peripheral lymph nodes appropriately, and are functional in presenting allogeneic and haptenic antigens. Moreover, T cells, as well as neutrophil and monocyte effector populations, are fully capable of entry into the inflamed skin sites in the absence of L-selectin. Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice. In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes. Indeed, L-selectin-deficient mice mount completely normal CHS responses when alternate routes of immunization are used. These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.

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L-selectin–deficient  mice have an intact antigen presentation pathway. (A) Epidermal whole mounts from L-selectin–deficient mice show normal  numbers and distribution of LC.  Whole mounts of abdominal  skin were prepared and stained  with anti-I-Ab (41). I-A+ cells  were counted in 30 fields/slide  (field = 0.1 mm2), and calculated  as the average of cells/mm2 over  two slides. (B) LC are present in  L-selectin–deficient mice and  drain to PLN following skin  painting with FITC. Mice were  sensitized with FITC and 24 h  later PLN and spleens were collected and centrifuged over metrizamide gradients (18). Cells at  the interface were collected and  stained with biotinylated anti– mouse I-A followed by streptavidin-PE and analyzed by twocolor FACS® analysis to detect  the presence of draining I-A+/ FITC+ LC. One of six representative experiments is shown. (C )  LC from L-selectin–deficient mice can initiate allogeneic responses. LC collected from PLN of FITC-painted wild-type (squares) or L-selectin–deficient  mice (triangles) were irradiated and incubated for 48 h with 2 × 105 T cells from either BALB/c (allogeneic; open squares and triangles) or C57BL/6J (syngeneic; closed squares and triangles) mice. Cultures were pulsed with [3H]thymidine for 18 h before harvesting. (D) LC can initiate antigen-specific responses. Irradiated PLN cells were used as APCs from wild-type or L-selectin–deficient mice, and were either pulsed with or without DNBS. These cells  were incubated for 48 h with increasing concentrations of PLN T cells isolated from wild-type mice sensitized with DNFB 5 d before. Cultures were  pulsed with [3H]thymidine for 18 h before harvesting.
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Figure 3: L-selectin–deficient mice have an intact antigen presentation pathway. (A) Epidermal whole mounts from L-selectin–deficient mice show normal numbers and distribution of LC. Whole mounts of abdominal skin were prepared and stained with anti-I-Ab (41). I-A+ cells were counted in 30 fields/slide (field = 0.1 mm2), and calculated as the average of cells/mm2 over two slides. (B) LC are present in L-selectin–deficient mice and drain to PLN following skin painting with FITC. Mice were sensitized with FITC and 24 h later PLN and spleens were collected and centrifuged over metrizamide gradients (18). Cells at the interface were collected and stained with biotinylated anti– mouse I-A followed by streptavidin-PE and analyzed by twocolor FACS® analysis to detect the presence of draining I-A+/ FITC+ LC. One of six representative experiments is shown. (C ) LC from L-selectin–deficient mice can initiate allogeneic responses. LC collected from PLN of FITC-painted wild-type (squares) or L-selectin–deficient mice (triangles) were irradiated and incubated for 48 h with 2 × 105 T cells from either BALB/c (allogeneic; open squares and triangles) or C57BL/6J (syngeneic; closed squares and triangles) mice. Cultures were pulsed with [3H]thymidine for 18 h before harvesting. (D) LC can initiate antigen-specific responses. Irradiated PLN cells were used as APCs from wild-type or L-selectin–deficient mice, and were either pulsed with or without DNBS. These cells were incubated for 48 h with increasing concentrations of PLN T cells isolated from wild-type mice sensitized with DNFB 5 d before. Cultures were pulsed with [3H]thymidine for 18 h before harvesting.

Mentions: It is known that LC are bone marrow derived (25) and that L-selectin is expressed on bone marrow progenitors (26). Therefore, their development and/or homing to skin may be impaired in these mice. Fig. 3 A shows a representative whole mount of abdominal skin from an L-selectin–deficient and wildtype mouse stained with anti-I-A. Comparison of L-selectin mutant mice and wild-type littermate controls showed that there was no difference in the density, morphology, or distribution of these cells. The average number of I-A+ epidermal cells in L-selectin–deficient mice was 1,050 ± 180/mm2 compared to 1,080 ± 160 /mm2 in wild-type mice. Thus, LC are present in normal densities in these L-selectin–deficient mice. In addition, no differences were noted in Thy-1+ dendritic epidermal T cells (data not shown).


The route of antigen entry determines the requirement for L-selectin during immune responses.

Catalina MD, Carroll MC, Arizpe H, Takashima A, Estess P, Siegelman MH - J. Exp. Med. (1996)

L-selectin–deficient  mice have an intact antigen presentation pathway. (A) Epidermal whole mounts from L-selectin–deficient mice show normal  numbers and distribution of LC.  Whole mounts of abdominal  skin were prepared and stained  with anti-I-Ab (41). I-A+ cells  were counted in 30 fields/slide  (field = 0.1 mm2), and calculated  as the average of cells/mm2 over  two slides. (B) LC are present in  L-selectin–deficient mice and  drain to PLN following skin  painting with FITC. Mice were  sensitized with FITC and 24 h  later PLN and spleens were collected and centrifuged over metrizamide gradients (18). Cells at  the interface were collected and  stained with biotinylated anti– mouse I-A followed by streptavidin-PE and analyzed by twocolor FACS® analysis to detect  the presence of draining I-A+/ FITC+ LC. One of six representative experiments is shown. (C )  LC from L-selectin–deficient mice can initiate allogeneic responses. LC collected from PLN of FITC-painted wild-type (squares) or L-selectin–deficient  mice (triangles) were irradiated and incubated for 48 h with 2 × 105 T cells from either BALB/c (allogeneic; open squares and triangles) or C57BL/6J (syngeneic; closed squares and triangles) mice. Cultures were pulsed with [3H]thymidine for 18 h before harvesting. (D) LC can initiate antigen-specific responses. Irradiated PLN cells were used as APCs from wild-type or L-selectin–deficient mice, and were either pulsed with or without DNBS. These cells  were incubated for 48 h with increasing concentrations of PLN T cells isolated from wild-type mice sensitized with DNFB 5 d before. Cultures were  pulsed with [3H]thymidine for 18 h before harvesting.
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Figure 3: L-selectin–deficient mice have an intact antigen presentation pathway. (A) Epidermal whole mounts from L-selectin–deficient mice show normal numbers and distribution of LC. Whole mounts of abdominal skin were prepared and stained with anti-I-Ab (41). I-A+ cells were counted in 30 fields/slide (field = 0.1 mm2), and calculated as the average of cells/mm2 over two slides. (B) LC are present in L-selectin–deficient mice and drain to PLN following skin painting with FITC. Mice were sensitized with FITC and 24 h later PLN and spleens were collected and centrifuged over metrizamide gradients (18). Cells at the interface were collected and stained with biotinylated anti– mouse I-A followed by streptavidin-PE and analyzed by twocolor FACS® analysis to detect the presence of draining I-A+/ FITC+ LC. One of six representative experiments is shown. (C ) LC from L-selectin–deficient mice can initiate allogeneic responses. LC collected from PLN of FITC-painted wild-type (squares) or L-selectin–deficient mice (triangles) were irradiated and incubated for 48 h with 2 × 105 T cells from either BALB/c (allogeneic; open squares and triangles) or C57BL/6J (syngeneic; closed squares and triangles) mice. Cultures were pulsed with [3H]thymidine for 18 h before harvesting. (D) LC can initiate antigen-specific responses. Irradiated PLN cells were used as APCs from wild-type or L-selectin–deficient mice, and were either pulsed with or without DNBS. These cells were incubated for 48 h with increasing concentrations of PLN T cells isolated from wild-type mice sensitized with DNFB 5 d before. Cultures were pulsed with [3H]thymidine for 18 h before harvesting.
Mentions: It is known that LC are bone marrow derived (25) and that L-selectin is expressed on bone marrow progenitors (26). Therefore, their development and/or homing to skin may be impaired in these mice. Fig. 3 A shows a representative whole mount of abdominal skin from an L-selectin–deficient and wildtype mouse stained with anti-I-A. Comparison of L-selectin mutant mice and wild-type littermate controls showed that there was no difference in the density, morphology, or distribution of these cells. The average number of I-A+ epidermal cells in L-selectin–deficient mice was 1,050 ± 180/mm2 compared to 1,080 ± 160 /mm2 in wild-type mice. Thus, LC are present in normal densities in these L-selectin–deficient mice. In addition, no differences were noted in Thy-1+ dendritic epidermal T cells (data not shown).

Bottom Line: Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice.In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes.These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235-9072, USA.

ABSTRACT
L-selectin, an adhesion molecule constitutively expressed on leukocytes, is important for primary adhesion and extravasation of lymphocytes at specialized high endothelial venules within lymph nodes and other leukocytes at sites of inflammation. We have generated L-selectin-deficient mice by targeted disruption, and have confirmed a previously reported phenotype which includes strikingly impaired contact hypersensitivity (CHS) responses to reactive haptens (Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. J. Exp. Med. 181:2259-2264; Xu, J.C., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. 183:589-598.). Since the mechanism of this impairment has not been clarified, we sought to define the stage(s) at which the CHS response is affected in L-selectin-deficient mice. We show that epidermal Langerhans cells in L-selectin-deficient mice are normal in number, migrate to peripheral lymph nodes appropriately, and are functional in presenting allogeneic and haptenic antigens. Moreover, T cells, as well as neutrophil and monocyte effector populations, are fully capable of entry into the inflamed skin sites in the absence of L-selectin. Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice. In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes. Indeed, L-selectin-deficient mice mount completely normal CHS responses when alternate routes of immunization are used. These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.

Show MeSH
Related in: MedlinePlus