Langerhans Cells Migrate to Local Lymph Nodes Following Cutaneous Infection with an Arbovirus  Linda J. Johnston, Nicholas J.C. King  Journal of Investigative Dermatology  Volume 114, Issue 3, Pages 560-568 (March 2000) DOI: 10.1046/j.1523-1747.2000.00904.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 FITC+ cell numbers are increased in lymph nodes draining virus-injected skin. DLN cells were collected 24 h (A) and 48 h (C) after intradermal injection with vehicle, SFV, WNV, or no injection and topical application of 1% FITC 18 h prior to being killed. Total cell numbers per local lymph node are shown for 24 h (B) and 48 h (D) after intradermal injection. Data shown are the mean ± SEM of four experiments, each making use of three or four animals. *p <0.05, **p <0.01 (Student’s t test compared with vehicle-injected animals). Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 (A) FITC+ cells in the lymph nodes draining virus-injected skin are NLDC145+ and MHC class II+. DLN cells collected from mice 18 h after topical application of 1% FITC and 24 h after injection with vehicle, SFV, or WNV were labeled with NLDC145 or MHC class II antibodies. Numbers in the right upper quadrant are the percentage of double-positive cells. Representative data are shown here and these data were similar in three replicate experiments, each making use of the amalgamated cells of three or four animals. (B) Gated high forward/high side scatter, FITC+ cells in the lymph nodes draining virus-injected skin are NLDC145+. DLN cells collected from mice 18 h after topical application of 1% FITC and 24 h after injection with vehicle, SFV, or WNV were labeled with NLDC145. Values in the right upper quadrant (FITC+/NLDC145+) in order from top to bottom are: absolute number of cells in the sample of 50,000 events, absolute percentage of cells and percentage of FITC+/NLDC145+ cells relative to the gated high forward/high side scatter population. Representative data are shown here and these data were similar in three replicate experiments, each making use of the amalgamated cells of three or four animals. Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Increased DC numbers in the DLN of virus-injected skin are independent of the application of FITC. Numbers of NLDC145+ cells per DLN were determined at 24 h (A) and 48 h (C) after intradermal injection with vehicle, SFV, or WNV. Total cell numbers per DLN are also shown at 24 h (B) and 48 h (D) after intradermal injection. Data shown are the mean ± SEM of four experiments, each making use of three or four animals. *p <0.05, **p <0.01 (Student’s t test compared with vehicle-injected animals). Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Langerhans cell morphology is altered in response to virus injection. Epidermal sheets were labeled with NLDC145 48 h after intradermal injection of vehicle (A) or SFV (B). Changes in morphology and density were similar if Langerhans cells were stained for MHC class II, as shown in epidermal sheets prepared from intradermal vehicle-injected (C) or WNV-injected (D) mice after 48 h. Scale bar: 10 μM. Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Langerhans cell density is decreased in the epidermis of virus-injected mice. NLDC145+ cell density in epidermal sheets at 24 h (A) or 48 h (B) after intradermal injection with vehicle, SFV, or WNV was determined by automated image analysis. Data shown are the mean ± SEM of three experiments, each making use of five animals. *p <0.05, **p <0.01 (Student’s t test compared with vehicle-injected animals). Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The Langerhans cell migratory response requires injection of live virus. DLN cells were collected at 24 h (A) and 48 h (C) after intradermal injection with vehicle, SFV, UV-inactivated SFV (UV-SFV), WNV, or UV-inactivated WNV (UV-WNV) and topical application of 1% FITC 18 h prior to sacrifice. Total cell numbers per local lymph node are shown for 24 h (B) and 48 h (D) after intradermal injection. Data shown are the mean ± SEM of four experiments, each making use of three or four animals. *p <0.05, **p <0.01, NS not significant (Student’s t test compared with vehicle-injected animals). Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 E-cadherin expression-positive cell numbers are altered during viral infection. Two-color immunofluorescence analysis of E-cadherin and MHC class II expression on epidermal cells 24 h (left-hand panels) and lymph node cells 48 h (right-hand panels) after injection with vehicle (B), SFV (C), WNV (D), UV-inactivated SFV (E), and UV-inactivated WNV (F). Immunoglobulin control versus MHC class II is shown in (A). Representative data are shown here and these data were similar in three replicate experiments, each making use of the amalgamated cells of three or four animals. Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 UV-inactivated WNV and SFV do not cause in vivo phenotypic changes in epidermal cells. Smoothed flow cytometric histograms of MHC-I-labeled epidermal cells are shown in the upper panels after 24 h WNV (A) and SFV (B) infection. Lower panels show zoomed detail in smoothed flow cytometric histograms of MHC class II-labeled epidermal cells, representing MHC class IIhi Langerhans cells, after 24 h WNV (C) and SFV (D). Mock-infected, live virus infected, UV-inactivated virus-infected and isotype control labeled epidermal cell histograms are shown as profiles a, b, c, and d, respectively, in all panels. Representative data are shown here and these data were similar in three replicate experiments, each making use of the amalgamated cells of three or four animals. Journal of Investigative Dermatology 2000 114, 560-568DOI: (10.1046/j.1523-1747.2000.00904.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions