Resident lymphocytes in the dermis of the normaldorsolateral thoracic skin of alpacas

Heather D. Edginton*, Jeanine Peters-Kennedy† and Danny W. Scott*

Departments of*Clinical Sciences and †Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

Correspondence: Danny W. Scott, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 USA.

E-mail: shb3@cornell.edu

Background – Small numbers of resident T lymphocytes are present in the dermis of normal skin of humans,

cattle and sheep.

Hypothesis/Objectives – Wewanted to determine the prevalence, numbers and immunophenotype of lympho-

cytes in the dermis of healthy skin from alpacas.

Animals – Skin biopsy specimens were collected from the dorsolateral thorax of 31 alpacas with normal skin.

Methods – Skin biopsy specimens were evaluated for the prevalence and numbers of CD3+ and CD79a+ lym-

phocytes.

Results – Resident CD3+ and CD79a+ lymphocytes were found around the superficial and deep dermal blood

vessels. The CD3+ lymphocytes were more numerous than CD79a+ lymphocytes. Both CD3+ and CD79a+ lym-

phocytes were more numerous around superficial dermal blood vessels.

Conclusions and clinical importance – Resident CD3+ and CD79a+ lymphocytes are present around superfi-

cial and deep dermal blood vessels in normal skin from alpacas; hence, the presence of lymphocytes in these

locations without obvious features of inflammation must be interpreted cautiously when evaluating skin biopsy

specimens from alpacas with skin disease.

Introduction

Alpacas have become increasingly popular in recent

years, thus creating the need for more knowledge about

their skin in health and disease. Recent publications have

characterized clinical, histopathological and therapeutic

aspects of skin diseases in alpacas,1 the microanatomy of

normal skin from alpacas2 and the resident lymphocyte

population in the epidermis and adnexal epithelia of nor-

mal skin from alpacas.3

The principal purpose of the skin immune system is to

provide protection from myriads of infectious agents (bac-

terial, fungal, viral, protozoal and parasitic).4 The skin

immune system is also involved with inflammation and

tissue repair. Key cellular components of the skin

immune system include keratinocytes, Langerhans cells,

dermal dendritic cells and resident intraepithelial and der-

mal T lymphocytes.4 Resident cutaneous intraepithelial

lymphocytes have been described in humans, mice, cat-

tle, sheep and alpacas (reviewed by Clark et al.).3 They

are of the T-lymphocyte immunophenotype and occur in

very small numbers.

Resident lymphocytes in the human dermis occur in

very small numbers and are predominately present around

blood vessels.5–10 Resident dermal lymphocytes in human

skin are CD3+ (pan T-cell marker) T lymphocytes.6–10 Res-

ident dermal CD3+ T lymphocytes have also been

described in the normal skin of cattle11 and sheep.12 In

contrast, B lymphocytes have not been found in the der-

mis of normal human skin.6–10 To the authors’ knowledge,

the occurrence of resident lymphocytes in the dermis of

normal alpaca skin has not been investigated.

Previous publications have documented the reliability

of immunophenotyping alpaca lymphocytes using antihu-

man CD3 (T lymphocytes) and CD79a (B lymphocytes

and plasma cells).3,13–15 The purpose of this study was to

determine the prevalence and numbers of CD3+ and

CD79a+ cells in the dermis of skin biopsy specimens

from 31 alpacas with normal skin.

Materials and methods

Archival samples of normal skin from 31 alpacas submit-

ted to the section of Anatomic Pathology for postmortem

examination from 2007 to 2011 were used in this study.

The alpacas ranged in age from <1 month to 16 years

and included 15 males and 16 females. The animals had

not received glucocorticoids within the 3 week period

prior to death. None of the animals had a recent history of

skin disease. All samples were taken from grossly normal

skin from the dorsolateral thorax using a 6 mm biopsy

punch. Samples were then formalin fixed and paraffin

embedded. Serial sections (4 lm thick) from each block

were stained with haematoxylin and eosin (H&E) and anti-

bodies against CD3 and CD79a. For the purposes of this

study, the term ‘sample’ will be used to define the slides

of one biopsy specimen.

Accepted 23 November 2013

Sources of Funding: This study was self-funded.

Conflict of Interest: No conflicts of interest have been declared.

© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 42–e15.42

Vet Dermatol 2014; 25: 42–e15 DOI: 10.1111/vde.12103

Histological evaluation

Sections stained with H&E (one section per alpaca) were

examined by two of the authors (H.D.E. and J.P.-K.). All

sections were examined by both authors. Each micro-

scopic field examined contained at least one vascular

plexus and was separated into superficial and deep der-

mis. The superficial dermis was defined as the region

extending from the epidermis to above the sebaceous

glands. The deep dermis was defined as the region

extending from below the sebaceous glands to the sub-

cutis. Three 940 microscopic fields (one at each end of

each specimen and one in the centre) were analysed for

the presence of CD3+ and CD79a+ superficial and deep

dermal lymphocytes.

Immunohistochemical evaluation

Immunohistochemistry for CD3 and CD79a was per-

formed as described in previous studies.3,13–15 Briefly,

sections were mounted and deparaffinized. The sections

were incubated with rabbit antihuman polyclonal CD3

antibody (Dako North America, Inc., Carpenteria, CA,

USA) at a 1:100 dilution and mouse antihuman monoclo-

nal CD79a antibody (Dako North America, Inc.) at a dilu-

tion of 1:20 and stained using a standardized technique.

The chromagen was 3,3′-diaminobenzidine tetrahydro-

chloride (DAB; Dakocytomation, Dako North America,

Inc.).

Normal alpaca lymph node and alpaca brain served as

positive and negative controls, respectively. Additionally,

diseased alpaca skin (bacterial folliculitis with numerous

dermal lymphocytes and plasma cells present on H&E)

was processed in an identical fashion to the samples in

question to serve as a positive tissue control. Diseased

alpaca skin also served as a negative tissue control, when

processed by substituting the primary antibody with non-

immune rabbit serum.

Results

All skin samples were histologically normal.2 Neutrophils,

eosinophils and macrophages were not seen. CD3+ lym-

phocytes were found in the superficial and deep dermis,

primarily in a perivascular location (Figure 1), in 31 of 31

samples (100%) and 27 of 31 samples (87%), respec-

tively (Table 1). CD79a+ lymphocytes were found in the

superficial and deep dermis, also in a perivascular location

(Figure 2), in 21 of 31 samples (68%) and 19 of 31 sam-

ples (61%), respectively. The total number of CD3+ lym-

phocytes (1426) counted in all samples was larger than

the total number of CD79a+ lymphocytes (261).

The total number of superficial perivascular CD3+ lym-

phocytes (1161) in all samples was larger than the total

number of deep perivascular CD3+ lymphocytes (265;

Table 1). The total number of superficial and deep peri-

vascular CD3+ lymphocytes counted per sample varied

from 1 to 106 (median 22) and from 0 to 41 (median 5),

respectively.

The total number of superficial perivascular CD79a+lymphocytes (175) in all samples was larger than the total

number of deep perivascular CD79a+ lymphocytes (86;

Table 1). The total number of superficial and deep peri-

vascular CD79a+ lymphocytes counted per sample varied

from 0 to 39 (median 1) and from 0 to 17 (median 1),

respectively. Given that alpaca B lymphocytes and

plasma cells are both CD79a+, the H&E samples were

scrutinized, and plasma cells were rarely seen (one or

two per sample). The CD79a+ cells did not have the

appearance of plasma cells.

Figure 1. Normal alpaca skin. Note CD3+ lymphocytes around

superficial dermal blood vessels (arrows). Scale bar represents

50 lm.

Table 1. The number of lymphocytes counted in the superficial and

deep dermis of normal alpaca skin*

Case

no.

Age

(months) Sex

No. of

CD3+superficial

No. of

CD3+deep

No. of

CD79a+superficial

No. of

CD79a+deep

1 24 F 90 3 2 1

2 24 M 15 10 1 0

3 7 F 51 25 0 3

4 54 M 22 20 0 0

5 48 F 18 2 0 0

6 72 M 32 3 1 1

7 132 F 106 28 39 4

8 Juvenile M 65 11 0 0

9 18 F 12 3 24 9

10 Adult M 21 7 0 0

11 72 F 5 6 4 11

12 8 M 85 15 2 1

13 3 F 93 18 16 12

14 7 F 42 7 26 17

15 10 F 61 10 21 2

16 24 F 73 41 0 0

17 12 M 13 6 10 2

18 24 M 68 12 5 6

19 6 F 55 5 1 0

20 13 M 9 0 0 1

21 16 M 15 5 0 0

22 5 F 3 0 1 0

23 12 F 16 2 1 1

24 36 F 10 1 0 0

25 18 M 46 15 1 1

26 11 M 18 3 0 2

27 120 M 52 2 6 0

28 11 M 8 4 2 1

29 36 M 49 1 8 9

30 168 F 7 0 1 0

31 192 F 1 0 3 2

Total 1161 265 175 86

Abbreviations: F, female; and M, male.

*Total number of cells counted in three 409microscopic fields.

© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 42–e15. 43

Resident dermal lymphocytes in alpacas

Discussion

Resident epidermal lymphocytes are a critical component

of the skin-associated lymphoid tissue or skin immune

system.6,16 Given that resident epidermal lymphocytes

are not known to replicate, they are presumably replaced

as needed by resident dermal lymphocytes. This notion is

supported by the fact that resident epidermal and dermal

lymphocytes in humans are of the same immunopheno-

type.6–10 Resident epidermal lymphocytes have previ-

ously been described in the alpaca3 and are of the same

CD3+ immunophenotype as the majority of resident der-

mal lymphocytes described in our study.

Resident dermal lymphocytes have previously been

described in humans, cattle and sheep. In humans, resi-

dent dermal T lymphocytes are present in very small

numbers (not quantified), primarily in association with

superficial dermal blood vessels.6–10 In sheep, resident

dermal T lymphocytes were found more commonly

around superficial dermal blood vessels (mean cell count

of 10 per 10 mm length of sample) than deep dermal

blood vessels (mean cell count of six per 10 mm length

of sample).12 In cattle, resident dermal T lymphocytes

were also found more commonly around superficial der-

mal blood vessels (cell count 166 � 27.4 per 15 cm 9

10 cm sample) than deep dermal blood vessels (cell

count 5.2 � 3.8 per 15 cm 9 10 cm sample).11

Resident dermal T lymphocytes were consistently

found in the alpaca samples evaluated. The number of

CD3+ T lymphocytes present in three 940 microscopic

fields within the superficial or deep dermis varied from 1

to 106 (median 22) and from 0 to 41 (median 5), respec-

tively. Due to differences in methodology (sample site

and size; manner of fixation and preparation), it is impossi-

ble to compare our results in alpacas with the ovine and

bovine data. However, it is clear that resident dermal

T lymphocytes occur in all three species, primarily in a

perivascular location and predominately around superficial

dermal blood vessels.

An unexpected finding in our study was the presence

of superficial and deep perivascular CD79a+ lymphocytes

in 68 and 61%, respectively, of the samples examined.

The CD79a+ lymphocytes were present in much smaller

numbers than CD3+ lymphocytes: 0–39 (median 1) and

0–17 (median 1) in three 940 microscopic fields within

the superficial or deep dermis, respectively. The CD79a+lymphocytes were also more numerous around superfi-

cial than deep dermal blood vessels. To our knowledge,

resident dermal B lymphocytes have not been described

in other species. The CD79a+ resident lymphocytes were

not found in the epidermis of healthy alpaca skin.3

Whether resident dermal B lymphocytes are unique to

alpacas is not known. Other regions of alpaca skin, as

well as skin from other camelids and other animal spe-

cies, would have to be investigated to make such a deter-

mination. Alternatively, previous antigenic challenge to

the dorsolateral thoracic skin (e.g. from ticks, lice or pour-

on medicaments) may have provoked an increase in der-

mal B-lymphocyte numbers. While the alpacas studied

had grossly normal skin and no recent history of skin dis-

ease, we cannot rule out a previous dorsally distributed

cutaneous insult. Again, other areas of alpaca skin would

need to be investigated.

In conclusion, resident CD3+ and CD79a+ lymphocytes

were found around the superficial and deep dermal blood

vessels in normal skin from alpacas. Therefore, the pres-

ence of lymphocytes in these locations without obvious

features of inflammation (such as vascular dilatation, con-

gestion and oedema) must be interpreted cautiously.

Acknowledgements

We thank the anatomic pathology residents for collecting

many of the normal alpaca skin specimens.

References

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Figure 2. Normal alpaca skin. Note CD79a+ lymphocytes around

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© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 42–e15.44

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R�esum�e

Contexte – De petits nombres de lymphocytes T r�esidents sont pr�esents dans le derme de la peau nor-

male de l’homme, des bovins et des moutons.

Hypoth�eses/Objectifs – Nous voulons d�eterminer la pr�evalence, le nombre et l’immunoph�enotype des

lymphocytes dans le derme de peau saine d’alpagas.

Sujets – Des biopsies cutan�ees de peu normale ont �et�e pr�elev�ees sur le thorax dorso-lat�eral de 31 alpagas.

M�ethodes – Les biopsies cutan�ees ont �et�e �evalu�ees pour la pr�evalence et le nombre de lymphocytes

CD3+ et de CD79a+.R�esultats – Les lymphocytes r�esidents CD3+ et CD79a+ ont �et�e trouv�es autour des vaisseaux sanguins

dermiques superficiels et profonds. Les lymphocytes CD3+ �etaient plus nombreux que les lymphocytes

CD79a+. Les lymphocytes CD3+ et CD79a+ �etaient plus nombreux autour des vaisseaux sanguins dermi-

ques superficiels.

Conclusions et importance clinique – Les lymphocytes r�esidents CD3+ et CD79a+ sont pr�esents autour

des vaisseaux sanguins dermiques superficiels et profonds de la peau normale d’alpagas; ainsi, la pr�esence

de lymphocytes dans cette localisation sans autre signe d’inflammation doit etre interpr�et�e avec pr�ecaution

au cours de lecture de biopsies cutan�ees d’alpagas avec une dermatose.

Resumen

Introducci�on – existe un peque~no numero de linfocitos residentes en la dermis de piel normal de hum-

anos, bovinos y ovinos.

Hip�otesis/Objetivos – nos propusimos determinar la prevalencia, n�umeros e inmunofenotipo de linfocitos

en la dermis de piel sana de alpacas.

Animales – se tomaron muestras de piel de la parte laterodorsal del tronco de 31 alpacas con piel normal.

M�etodos – se evaluaron las muestras de piel para prevalencia y n�umeros de linfocitos CD3+ y CD79a+.Resultados – se encontraron linfocitos residentes CD3+ y CD79a+ alrededor de los vasos superficiales y

profundos de la dermis. Los linfocitos CD3+ fueron mas numerosos de los linfocitos CD79a+. Ambos tipos

de linfocitos CD3+ y CD79a+ fueron m�as numerosos alrededor de los vasos d�ermicos superficiales.

Conclusiones e importancia cl�ınica – Los linfocitos residentes CD3+ y CD79a+ est�an presentes alrede-

dor de los vasos superficiales y profundos de la dermis en la piel normal de alpacas; por lo tanto la presen-

cia de linfocitos en estas zonas sin caracter�ısticas de inflamaci�on se debe interpretar con cautela cuando se

eval�uen biopsias de piel de alpacas con enfermedades de la piel

Zusammenfassung

Hintergrund – Eine kleine Anzahl von residenten T Lymphozyten sind in der Dermis normaler Haut des

Menschen, Rindes und Schafes vorhanden.

Hypothese/Ziele – Wir wollten das Vorkommen, die Anzahl und den Immunph€anotyp der Lymphozyten in

der Dermis gesunder Alpakahaut bestimmen.

Tiere – Es wurden Hautbiopsieproben vom dorsolateralen Thorax von 31 Alpakas mit normaler Haut entn-

ommen.

Methoden – Die Hautbiopsieproben wurden auf das Vorkommen und auf die Anzahl von CD3+ und

CD79a+ Lymphozyten hin untersucht.

Ergebnisse – Residente CD3+ und CD79a+ Lymphozyten wurden im Bereich der oberfl€achlichen und tie-

fen dermalen Blutgef€aße gefunden. Die CD3+ Lymphozyten waren zahlreicher vorhanden als CD79a+Lymphozyten. Sowohl CD3+ als auch CD79a+ Lymphozyten kamen rund um die oberfl€achlichen dermalen

Blutgef€aße h€aufiger vor.

Schlussfolgerungen und klinische Bedeutung – Residente CD3+ und CD79a+ Lymphzyten sind rund

um oberfl€achliche und tiefe dermale Blutgef€aße in der normalen Alpakahaut vorhanden; daher muss das

Auftreten von Lymphozyten in dieser Lokalisation ohne offensichtliche Anzeichen einer Entz€undung vors-

ichtig interpretiert werden, wenn Hautbiopsieproben von Alpakas mit einer Hauterkrankung evaluiert wer-

den.

© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 42–e15. 45

Resident dermal lymphocytes in alpacas

Edginton et al.

© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 42–e15.e15