The cutaneous DTH response to antigen is widely considered to be a manifestation of memory T cell responsiveness in vivo. The central observation of this study is that this response to challenge by a panel of recall antigens was defective in older humans. In addition, the incidence of positive contact hypersensitivity reactions is also decreased in the elderly (Piaserico et al., 2004
; Balato et al., 2008
). This suggests that there may be a generalized defect in skin reactivity during aging. This may be the biological basis for the increased susceptibility of old subjects to both cutaneous malignancy and infections (Laube, 2004
; Diffey and Langtry, 2005
). An unexpected observation was that the defect in the response to cutaneous antigenic challenge was not in the memory T cell compartment, but in the innate conditioning of the skin after antigen stimulation.
Although T cell numbers are decreased after antigen challenge, macrophages identified by CD163 (this study) or CD68 and CD14 (unpublished data) are present in equal numbers in young and old skin. This is likely because of the different trafficking requirements of different leukocytes. It has been shown that DC-SIGN–ICAM-2 interactions mediate dendritic cell trafficking (Geijtenbeek et al., 2000
), and DC-SIGN is also strongly expressed on CD163+
macrophages in both normal and inflamed skin (Zaba et al., 2007
). We have also found high levels of DC-SIGN expression by activated peripheral blood monocytes (unpublished data). ICAM-2 is constitutively expressed on endothelial cells and, in contrast to E-selectin, VCAM-1, and ICAM-1, its expression is not dependent on the presence of TNF-α (Silverman et al., 2001
). Activated monocytes may therefore enter the skin by alternative pathways of transmigration through the endothelium compared with memory T cells. Furthermore, significant numbers of CD163+
macrophages are present in the skin of both young and old subjects, even without injection of antigen; therefore, the recruitment of circulating myeloid cells at the very early stages of the response may not be a limiting step in DTH responses of old humans.
The dominant role of TNF-α in regulating leukocyte migration via the activation of endothelial cells is supported by studies using models of contact hypersensitivity (McHale et al., 1999
). Furthermore, it was found that anti–TNF-α therapy in humans decreases the expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-1 in various assays, leading to reduced trafficking of leukocytes (Paleolog et al., 1998
). This decreased endothelial activation is associated with an increase in the frequency of skin and soft tissue infections in anti–TNF-α–treated rheumatoid arthritis patients compared with those receiving traditional disease-modifying antirheumatic drugs (Dixon et al., 2006
The need for appropriate leukocyte migration for immunosurveillance and the prevention of disease is further underscored by the clinical use of anti-α4β1 integrin mAb that binds to VLA-4, the main homing molecule involved in lymphocyte migration to inflamed brain in rodent models of multiple sclerosis (Engelhardt et al., 1995
). A mAb to this molecule reduced the inflammatory infiltrate in brain tissue and blocked clinical paralysis in various animal models of multiple sclerosis (Yednock et al., 1992
; Theien et al., 2003
; Deloire et al., 2004
). However, the use of Natalizumab, which is a humanized mAb to α4β1 integrin, for treatment of multiple sclerosis led to the development of severe opportunistic brain infections (Steinman, 2005
). Thus, inhibiting leukocyte migration in humans can lead to severe pathological consequences. The defect in macrophage triggering to secrete TNF-α in the skin raises the question of whether there is a defect in endothelial activation during immune responses in other organs during aging, as the potential decrease in T cell immunosurveillance may contribute to the reported increase in susceptibility of old individuals to a wide range of infections (Nicholson et al., 1997
; Yoshikawa, 2000
; Schmader, 2001
An important unanswered question is why macrophages in the old skin are not triggered to secrete TNF-α. Previous studies have shown that there is an age associated defect in human TLR1 and 2 function that results in a significant defect in TNF-α secretion after ligation of these receptors (van Duin et al., 2007
). Because C. albicans
activates TLR1 and 2, this may explain the cutaneous defect in macrophage-derived TNF-α that we have observed. It has also been shown that there is a defect in TLR4 function and expression and function during aging (Renshaw et al., 2002
; Krabbe et al., 2004
; van den Biggelaar et al., 2004
), although this has not been confirmed in other studies (van Duin and Shaw, 2007
). However, we found that both peripheral blood monocytes and isolated cutaneous macrophages from donors of both age groups synthesized similar levels of TNF-α after TLR-1/2 and TLR-4 stimulation in vitro. We therefore conclude that cutaneous macrophages in old humans are not inherently defective in terms of TNF-α synthesis, but do not rule out the possibility that other functions in these cells may be altered during aging. This is currently under investigation.
In addition to the need for TLR signaling for macrophage activation, these cells must also interact with cytokines such as IFN-γ to become fully functional. Previous studies have shown that IFN-γ is mainly secreted by T cells during DTH responses (Chu et al., 1992
). The lack of IFN-γ in the skin in our studies may reflect the decreased infiltration of T cells into the site of antigen challenge. Activation by IFN-γ leads to the expression of several genes that regulate macrophage biology, including expression of MHC class II genes (Celada and Maki, 1991
; Goñalons et al., 1998
; Cullell-Young et al., 2001
), which are crucial for antigen presentation to T lymphocytes. It is possible that the lack of T cell signals such as IFN-γ early in the DTH response in old subjects may prevent the cascade of events required for amplification of the response.
An alternative possibility is that macrophages in the skin of old individuals may be inhibited functionally in situ. It has been shown that human CD4+
T reg cells are potent inhibitors of macrophage activation and TNF-α secretion by these cells (Taams et al., 2005
; Tiemessen et al., 2007
). In addition, in a previous study we showed that there is a significant increase in functional circulating T reg cells in old individuals (Vukmanovic-Stejic et al., 2006
), which is in agreement with the observation that functional T reg cells accumulate in tissues of aged mice (Lages et al., 2008
). We now show that there are significantly higher proportions of CD4+
T cells in the skin of old volunteers either before or 7 d after injection of C. albicans
antigens. Although circumstantial, this suggests that the accumulation of T reg cells in the skin may be one possible mechanism for the observed decrease of TNF-α secretion by cutaneous macrophages in old individuals. This is currently under investigation.
With increasing age, cellular debris is not cleared optimally and accumulates within blood vessels and tissues, triggering nonspecific immune responses (for review see Richards et al., 2007
). This may manifest as atherosclerosis, the accumulation of amyloid proteins leading to Alzheimer's disease, lipofuscin pigments leading to age-related macular degeneration, and crystal arthritis that results from the defective removal of uric acid (Richards et al., 2007
). The reduced activation of macrophages in the skin and possibly other tissues may reduce the occurrence of chronic nonspecific inflammatory responses in old individuals. However, this reduced activation can also lead to decreased tissue-specific immunosurveillance by memory T cells. For successful aging to occur, it is therefore crucial to reduce nonspecific inflammation, yet retain sufficient protective immunity. The regulation of this homeostatic status quo clearly requires further investigation.