PMC

Quantification of cells expressing IP-10 and MIG mRNA during elicitation of CHS. In situ hybridization for these chemokines was performed as described in Materials and Methods. Results are expressed as mean percentage of specific mRNA-expressing cells per counting field of four to seven sections (±SEM) at each time point until 96 hours after allergen exposure.

Quantification of cells expressing LARC, PARC, TARC, MDC mRNA during the course of CHS. In situ hybridization for these chemokines was performed as described in Materials and Methods. Results are expressed as mean percentage of specific mRNA-expressing dermal cells per counting field of four to seven sections (±SEM) at each time point until 96 hours after elicitation of CHS.

Expression of MCP-1 and MDC 72 hours after elicitation of CHS. In situ hybridization was performed with anti-sense probes for MCP-1 (A and B) and MDC (C and D) on serial sections. In contrast to MCP-1, MDC is mainly expressed by inflammatory cells of the dermis and by a few cells invading the epidermis. Illumination: bright field (A and C), dark field (B and D). Objective, ×10.

Quantification of cells expressing MCP-1 (filled circle), RANTES (open circle), MIP-1α (filled square) and MIP-1β (open square) mRNA after elicitation of CHS. In situ hybridization for these chemokines was performed as described in Materials and Methods. Results are expressed as mean (±SEM) percentage of specific mRNA-expressing dermal cells per counting field of four to seven sections at each time point until 96 hours after elicitation of CHS.

Pronounced expression of IP-10, MIG, and MCP-1 mRNA 48 hours after elicitation of CHS. Serial skin sections were processed for in situ hybridization using ³⁵S-UTP-labeled anti-sense probes of IP-10 (A and B), MIG (C and D), and MCP-1 (E and F). Simultaneous IP-10, MIG, and MCP-1 expression is co-localized in the basal epidermal layer; notably, IP-10 and MIG mRNA is also strongly expressed by suprabasal cells. Illumination: bright field (A, C, and E), dark field (B, D, and F). Objective, ×10.

Differential expression of chemokines MCP-1, MIG, PARC, and TARC 48 hours after elicitation of CHS. In situ hybridization was performed with ³⁵S-UTP-labeled anti-sense probes of MCP-1 (A and B), MIG (C and D), PARC (E and F), and TARC (G and H) using serial sections. MIG mRNA signals are focally concentrated in the epidermis (C, arrows) whereas PARC and TARC messages are exclusively expressed below in the dermal compartment. Illumination: bright field (A, C, E, and G), dark field (B, D, F, and H). Objective, ×10.

Expression of macrophage-attracting chemokines MCP-1, RANTES, and MIP-1α 48 hours after elicitation of CHS. In situ hybridization with anti-sense probes against MCP-1 (A and B), RANTES (C and D), and MIP-1α (E and F) was performed on serial skin sections to compare relative intensity and localization of chemokine message. Both MCP-1 and RANTES are preferentially expressed in lesional basal keratinocytes with few positive cells in the upper dermis. MIP-1α mRNA+ cells are sparse. Illumination: bright field (A, C, and E), dark field (B, D, and F). Objective, ×20.

Spatial and temporal correlation between chemokine expression and recruitment of corresponding target cells. Sections obtained 24 hours after elicitation of CHS were hybridized with radioactively labeled anti-sense probes of MCP-1 (A and B) and MIG (D and E). Corresponding serial sections were immunohistologically labeled with macrophage (anti-CD68) (C) or T-lymphocyte-specific (anti-CD3) (F) mAbs as described in Materials and Methods. Expression of MCP-1 (A and B) and MIG (D and E) is overlapping with areas of macrophage (C) and T-lymphocyte (F) infiltration, respectively. Illumination: bright field (A, C, D, and F), dark field (B and E). Objective, ×10.

MCP-1 expression during elicitation of CHS. Biopsies obtained at 6 hours (A), 12 hours (B), 24 hours (C), and 48 hours (D) after epicutaneous application of hapten were processed for in situ hybridization using a MCP-1 anti-sense probe as described in Materials and Methods. The arrows point to focal expression of MCP-1 mRNA in the basal epidermal layer at 6 hours (A). At 12 hours, there is a nearly continuous expression and at 24 and 48 hours a strong continuous expression in the basal layer. At 24 and 48 hours, strong MCP-1 message is also detectable in the dermal compartment in a perivascular distribution at sites of leukocyte accumulation (C and D). Dark-field illumination. Objective, ×10.

Time course of leukocyte recruitment into the dermis during elicitation of CHS. Inflammatory cells were identified by immunostaining with mAbs against CD3 (T lymphocytes, filled circle), CD68 (macrophages, open circle) and neutrophil elastase (NE) (neutrophils, filled square). CHS was elicited as described in Materials and Methods and biopsies obtained at the time intervals indicated. Results are presented as percentage ± SEM of stained cells related to the total number of cells of at least three randomly selected dermal areas. For each time point, four to seven sections were evaluated (for details, see Materials and Methods).

Multistep navigation of leukocyte trafficking during elicitation of CHS. The schematic drawing shows the time course and the spatial distribution of chemokine expression and leukocyte recruitment. During the early phase of CHS (<12 hours after allergen exposure) only one chemokine, MCP-1, is induced and expressed exclusively by single basal keratinocytes. In the intermediate phase (12 to 48 hours after allergen exposure) increasing strong dermal and particularly epidermal expression of MCP-1 and RANTES as well as dermal expression of MDC, PARC, and TARC is paralleled by pronounced recruitment of monocytes and T lymphocytes to the dermal compartment. During the late phase (48 to 96 hours after allergen exposure) expression of high levels of IP-10 and MIG in basal and suprabasal epidermal layers is associated with recruitment of lymphocytes to the epidermis. Monocytes reside in the dermis, which may be because of a lack of expression of monocyte-attractant chemokines in suprabasal layers of the epidermis. Arrowheads point to the maximum of each chemokine gradient.