MARK D. FARRAR, PhD EILEEN INGHAM, PhD
Abstract: The inflammatory stage of acne vulgaris is usually of greatest concern to the patient. A number of morphologically different inflammatory lesions may form that can be painful and unsightly. In 30% of patients, such lesions lead to scarring.1 Inflammatory acne and acne scarring can have significant psychological effects on the patient, including depression, anxiety, and poor self-image.2Although inflammatory acne has been well characterized clinically, the mechanisms by which inflammatory lesions arise are still poorly understood. The human skin commensal bacterium,Propionibacterium acnes, has long been associated with inflammatory acne. This organism has been implicated over and above all of the other cutaneous microflora in contributing to the inflammatory response characteristic of acne. However, its precise role in the disease and its interaction with the human immune system remain to be elucidated.
P. acnesand Acne
P.
acneshas been implicated in the pathogenesis of acne for more than 100 years. It was initially believed to be the direct cause of the disease when it was first isolated in 1896, and subsequent stud-ies demonstrating an inflammatory response after in-jection ofP. acnes into the skin reinforced this view.3,4 However, these findings were cast into doubt when the organism was shown to reside on normal human skin 50 years later.5 The association between P. acnes and acne has been reaffirmed over the past 30 years through the use of antibiotics for acne treatment. Initial studies showed that antibiotic treatment of acne was therapeu-tic and resulted in a decrease in the population density ofP. acneson the skin.6However, antibiotics are known to be able to modulate the human immune system and thus may have a more direct anti-inflammatory activi-ty.7 The strongest evidence to date for an association betweenP. acnes and inflammatory acne came from a study that found erythromycin treatment of acne pa-tients to be therapeutic and that failure of this therapy in a number of individuals was associated with the development of erythromycin resistance inP. acnes.8It is now believed thatP. acnesis not the cause of acne, but is a significant contributing factor to the inflammatory stages of the disease.Development of Inflammatory Lesions: Comedogenesis
Inflammatory lesions develop from comedones arising due to an abnormal pattern of keratinization in the sebaceous follicle.1Comedogenesis was once thought to
not be part of the inflammatory process, but detailed immunohistological studies that have followed the de-velopment of inflammatory acne lesions have shown this to be the first step toward formation of an inflam-matory lesion. The microcomedone is now believed to be the earliest type of subclinical acne lesion.
Microcomedones develop into full comedones, which may be either open (“blackhead”) or closed (“whitehead”), and inflammatory lesions. Several dif-ferent types of inflammatory lesion may arise that can be distinguished clinically. Papules and pustules may be up to 5 mm in size with a raised area of erythema. Nodules and cysts are larger lesions that are associated with more severe acne. Nodules may have an inflam-matory area of 5 mm or more and persist for several weeks. Cysts may be as large as a few centimeters in size; it is these lesions that often lead to scarring. The resolving stage of an acne lesion is the macule, which is seen clinically as an area of erythema.9
During comedogenesis, two changes in the normal pattern of keratinization occur: (1) hyperproliferation of keratinocytes lining the follicle wall, as shown by an increase in the cell proliferation marker Ki-67, and (2) reduced desquamation due to increased cohesion be-tween keratinocytes.10 –12 These changes lead to an ac-cumulation of cornified keratinocytes within the folli-cle. It has been proposed that comedogenesis is a result of changes in the rate of secretion and composition of sebum, most notably a decrease in the concentration of linoleic acid.13,14 This process may involve cytokines. High levels of biologically active interleukin (IL)-1␣ have been demonstrated in comedones.15 This proin-flammatory cytokine has been shown to induce hyperk-eratinization in isolated sections of the human seba-ceous follicle wall (infrainfundibulum) in vitro.16
The role of P. acnes in comedogenesis is uncertain. Formalin-killed P. acnes cells do not induce normal human keratinocytes to produce IL-1␣ in vitro.17,18 However, a more recent study in our laboratory has
From the Skin Research Centre, Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK.
Address correspondence to Eileen Ingham, PhD, Division of Microbiol-ogy, School of Biochemistry and Molecular BiolMicrobiol-ogy, University of Leeds, Leeds LS2 9JT, UK.
E-mail address: E.Ingham@leeds.ac.uk
© 2004 by Elsevier Inc. All rights reserved. 0738-081X/04/$–see front matter 360 Park Avenue South, New York, NY 10010 doi:10.1016/j.clindermatol.2004.03.006
demonstrated that viable P. acnes is able to induce human keratinocytes to produce IL-1␣, tumor necrosis factor (TNF)-␣, and granulocyte-macrophage colony– stimulating factor (GM-CSF).19 If this process is mir-rored in vivo, thenP. acneswithin the sebaceous follicle may contribute to the process of comedogenesis through IL-1␣–induced hyperkeratinization. Interest-ingly, this study demonstrated variations in the stimu-lation of keratinocytes by P. acnes in different growth phases, with cells in the stationary phase inducing higher levels of cytokines than those in the exponential phase. This may be significant in vivo, because there may be differences in the microenvironment of different sebaceous follicles resulting in differentP. acnesgrowth phases. This may explain why in acne, only a propor-tion of follicles are affected at any one time.
Inflammation: Sequence of Events
Debate over the initial cellular infiltrate in inflamma-tory acne lesions has continued since the first histolog-ical studies were carried out 30 – 40 years ago. Results of some of the first studies concluded that the lymphocyte was the initiating inflammatory cell type.20 –22 How-ever, Kligman’s 1974 study10 concluded that there was an initial infiltrate of neutrophils followed by micro-scopic rupture of the sebaceous follicle wall and subse-quent formation of a clinically visible inflamed lesion. This course of events is still described in much of the acne literature as the process by which inflamed lesions arise, mediated by neutrophil chemoattractants pro-duced by P. acnes that diffuse through the follicle wall.23,24 It has been suggested that inflammation in acne results from a type IV hypersensitivity reaction to P. acnes or other comedonal components after the re-lease of reactive oxygen radicals and enzymes by neu-trophils and rupture of the follicle wall.16,23–25
Although it is widely believed that neutrophils are the initial cells infiltrating acne lesions and that these cells initiate inflammation, there is little evidence to support this. More recent histological studies have pro-vided compelling evidence that T lymphocytes are in-volved in the initiation of inflammation. A 1988 study used lesion mapping to accurately determine the age of inflammatory lesions.26 This study demonstrated that the initial infiltrate of all developing inflammatory le-sions examined consisted of mononuclear cells that were predominantly CD4⫹ T cells. Neutrophils were seen later in the course of inflammation, being detected in 33% of lesions of 72 hours’ duration. These observa-tions were confirmed in a later study that found CD4⫹ T cells present around lesions of less than 6 hours’ duration with no disruption of the follicle wall.27 Neu-trophils were seen in only a small proportion of in-flamed follicles of greater than 24 hours’ duration. In addition to characterizing the cellular infiltrate, this
study also investigated the expression of various cell surface molecules on cells of the infiltrate and sur-rounding tissue. Vascular expression of intercellular adhesion molecule-1 (ICAM-1), E-selectin, vascular cell adhesion molecule-1, and human leukocyte–associated antigen-Dr (HLA-Dr) was observed, as was expression of ICAM-1 and HLA-Dr by cells of the infiltrate and expression of ICAM-1 by follicle wall basal keratino-cytes.
An extension to these observations was made in a more recent study.28 Biopsy specimens were obtained from normal sebaceous follicles from individuals with-out acne, noninflamed follicles from acne patients, and inflamed papules of less than 6 hours’ duration. Biopsy sections were stained for inflammatory cells, vascular and proliferative markers, integrins, and the proinflam-matory cytokine IL-1␣. Large numbers of CD4⫹T cells (most of which were memory/effector cells) and mac-rophages, were seen around uninvolved follicles from acne patients, whereas none were found in non-acne controls. In addition, there was a lack of neutrophils and a reduction in the number of Langerhans cells in the perifollicular epidermis compared with controls. These features are characteristic of a specific immune response. Interestingly, there was an absence of kera-tinocyte hyperproliferation, and follicles did not dis-play any of the features of microcomedones, suggesting that these inflammatory events are not simply second-ary to hypercornification, but may be the earliest events in acne lesion formation. The results of these studies all point toward inflammation being initiated by CD4⫹T cells and not neutrophils. This would of course suggest that the initiation of inflammation in acne is in response to a specific antigen, as opposed to a nonspecific innate response to comedonal components.
Later events in the development of inflammatory lesions include the infiltration of neutrophils and pos-sibly the disruption of the follicle wall. Finally, the inflammatory response is down-regulated, allowing re-pair of the follicle through normal wound-healing mechanisms. In about 1/3 of individuals, healing of inflamed lesions leads to scarring.1 The mechanisms behind this process remain unclear, however. The se-quence of events in the formation of inflammatory acne lesions is summarized inFigure 1.
The Role ofP. acnesin Inflammation
P. acneshas been shown to be associated with inflam-matory acne through antibiotic resistance studies.6,8 However, this bacterium has not been shown to be a direct cause of the disease or to be involved in the initiation of inflammation. Numbers of viable bacteria within follicles show no correlation with severity of inflammation, and some inflamed lesions do not con-tain viableP. acnes.29However, nonviableP. acnescells
are immunostimulatory.30 P. acnesproduces a number of enzymes and biologically active molecules, some of which have chemoattractant activity, and it has also been shown to stimulate cells of the nonspecific im-mune system to produce proinflammatory cytokines, for example, the induction of TNF-␣, IL-1, and IL-8 production by monocytes.31,32
Recently, P. acnes has been shown to have T-cell mitogenic activity.33 Such activity could contribute to inflammation in acne through the activation of T cells and the release of immunological cytokines. It has been suggested that toll-like receptors (TLRs) may contribute to inflammation in acne through the activation of TLR2 on macrophages by P. acnes.34 Activation of TLR on monocytes leads to the release of proinflammatory cy-tokines IL-12 and IL-8, the latter of which is a neutro-phil chemoattractant. TLR2 and TLR4 are expressed on
human keratinocytes, and activation leads to IL-8 pro-duction.35,36A recent review of innate immunity in acne discussed various mechanisms of innate immunity and the role of these mechanisms in inflammation.37 It has been suggested thatP. acnesmay contribute to inflam-mation in acne through activation of TLRs expressed on keratinocytes and sebocytes, which in turn would lead to release of proinflammatory cytokines.
In intact follicles,P. acneswill not be in contact with T cells or macrophages; therefore, the aforementioned processes involving activation of these cells byP. acnes are likely to be significant only if rupture of the follicle occurs. Therefore, the direct effects ofP. acneson T cells and macrophages is likely to augment the established immune response rather than initiate it. However, di-rect contact betweenP. acnes and viable keratinocytes could occur deep within the follicle where the layer of cornified cells is extremely thin (only two or three cells thick) and extremely fragile.10 It is here that the inter-action betweenP. acnesand keratinocytes leading to the release of proinflammatory cytokines is more likely to occur.
Although it has been shown thatP. acnescan activate different arms of the innate immune system, this does not explain why at any one time only a proportion of sebaceous follicles are inflamed and P. acnes can be isolated from unaffected skin and follicles. More impor-tant, it does not provide an explanation for the resolu-tion of inflammatory acne. Spontaneous resoluresolu-tion of the disease occurs in most cases when individuals reach their late teens or early twenties. This resolution is not accompanied by a reduction in either the population density ofP. acneson the skin or in the sebum excretion rate, both of which increase at puberty, when the onset of acne is most common.1,23
Hypotheses and Perspectives
The spontaneous resolution of acne is one of the most intriguing features of the disease, and any hypothesis concerning the pathogenesis of acne must attempt to provide an explanation for this event. There is now strong evidence that inflammation in acne is initiated by a specific CD4⫹ T-cell response. The lymphocytic infiltrate observed in early inflammatory lesions could be due to specific recruitment of T cells. Changes in the follicle microenvironment may lead to an increase in the population density of P. acnes within the follicle, with increased production of immunogenic proteins. Such antigens would be processed by Langerhans cells present in the follicle wall. These cells would also be stimulated by the cytokines produced by keratinocytes in response to P. acnes, namely TNF-␣ and GM-CSF. The stimulated Langerhans cells would migrate via the afferent lymph vessels to the local lymph node, where antigens would be presented to CD4⫹ T cells.
Subse-Figure 1. Events in the evolution of an inflammatory acne lesion. The normal sebaceous follicle develops into a microcomedone and a comedone through keratinocyte hyperproliferation and reduced sloughing. This process is associated with an increase in levels of IL-1␣.P. acnes population density increases. Initiation of inflammation may occur through either a specific CD4⫹T-cell–mediated pathway or a nonspecific pathway involving increased production of
proinflammatory cytokines by keratinocytes. Inflammation is augmented by activation of macrophages and neutrophils. Finally, the inflammatory response is down-regulated, allowing the lesion to heal.
quent activation of these T cells would cause them to migrate to the skin via the efferent lymphatic vessels. At the sebaceous follicle, release of cytokines by these T cells and activation of mononuclear cells would lead to disruption of the follicle and visible inflammation. Clearance of the antigenic stimulus would allow the inflammatory response to be down-regulated and en-able the lesion to heal.
Conversely, the initial events may be nonspecific and followed by an antigenic-specific response, as was pro-posed by Holland et al.38In this scenario, the increase in sebum production at puberty in certain follicles would lead to a deficiency of linoleic acid within the follicle. An increase in water activity within the follicle due to perturbed barrier function would promote the growth ofP. acnes, and the population density would increase. It has been shown thatP. acnes can induce IL-1␣ pro-duction by keratinocytes. This may occur deep within the follicle, where the keratinized layers of cells are thinnest andP. acnesmay come into contact with viable keratinocytes. In addition, disruption of keratinocytes by extracellular products ofP. acnescould also lead to release of IL-1␣. Diffusion of IL-1␣into the surrounding dermis would cause the nonspecific activation of mono-nuclear and endothelial cells, leading to inflammation and disruption of the follicle. Release of P. acnes anti-gens into the dermis may then further exacerbate in-flammation through activation of CD4⫹T cells, as de-scribed earlier.
This cycle of inflammation and resolution of individ-ual follicles may explain the clinical appearance and healing of acne lesions, but it does not explain the complete resolution of the disease. An explanation for this phenomenon may lie with the role of P. acnes in inflammatory acne. The immune response toP. acnesof normal individuals and those suffering from acne has been well characterized. Sensitization to P. acnes has been demonstrated in normal individuals of all ages as well as in acne patients.1It has been shown that anti-body responses toP. acnesdiffer between normal indi-viduals and acne patients. Two-dimensional polyacryl-amide gel electrophoresis studies have shown that antibodies from acne patients react to a wider range of P. acnesprotein antigens than those from normal con-trols.39It should be noted, however, that this increased response toP. acnesmay be due to increased exposure to the bacterium as a consequence of the disease. Our current hypothesis on the role of P. acnes in acne is focused on heat-shock proteins (HSPs), which are highly immunogenic. The increase in population den-sity ofP. acnesat puberty may put the organisms under nutritional stress in a proportion of follicles. In response to this stress, the organisms may increase production of HSPs, which then activate CD4⫹ T cells via presenta-tion on Langerhans cells. Over time, this response may become negatively regulated due to the high degree of
homology between bacterial and human HSPs and the potential for autoreactivity. This hypothesis allows for the initiation of inflammation by either an antigen-specific mechanism or a non–antigen-antigen-specific mecha-nism and also offers an explanation for the resolution of acne. To facilitate studies in this area, the genes encod-ing the two main HSPs ofP. acnes, HSP60 (GroEL) and HSP70 (DnaK), have been cloned and sequenced, and the recombinant proteins have been produced in Esch-erichia coli and purified.40 Initial immunologic studies
have demonstrated the ability of these proteins to in-duce proinflammatory cytokine production by human keratinocytes in vitro.19Future studies should focus on determining whether these proteins play a role in in-flammatory acne.
Inflammatory acne and its link toP. acnesremains a much underresearched area of cutaneous biology. The precise role of P. acnes in acne and the inflammatory mechanisms involved have yet to be fully elucidated. Further studies into the interaction between the human immune system and P. acnes will hopefully lead to a greater understanding of the pathogenesis of acne and in turn lead to the development of improved treatments for this common disease. There is also a need for more extensive investigations into the more general role of the skin in immunity. How the skin’s immune system is modulated by and interacts with commensal skin bac-teria is a crucial question in understanding the immune mechanisms that operate in this extremely important arm of the human immune system.
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