Genitourin Med 1988;64:289-93 Infiltrate of syphilitic lesions before and after treatment A TOSCA, J LEHOU, M HATJIVASILIOU, A VARELZIDIS, JD STRATIGOS From the Department of Dermatology, "Andreas Sygros" Hospital, School of Medicine, National University of Athens, Athens, Greece sumMARY An immunohistological study of skin biopsy specimens from patients with early syphilis was undertaken before and after treatment (one day after intramuscular administration of 2.4 MIU benzathine penicillin and eight days later, after a total administration of 3.6 MIU. In chancres from seronegative patients treatment with 3.6 MIU usually resulted in fewer immunocompetent cells in the infiltrate. In lesions of secondary syphilis treatment with 2.4 MIU benzathine penicillin produced a significant decrease in immunocompetent cells. After treatment with 3.6 MIU there was no further decrease. It was worth noticing that even eight to nine days after the initial pretreatment biopsy, when 3.6 MIU had been administered, the overall lymphohistiocytic infiltrate was not substantially diminished. Significantly more suppressor (T8+) cells were found in lesions of primary syphilis than of secondary syphilis, and they showed remarkable exocytosis. Activated local T8+ cells may release immunosuppressive lymphokines. Abundant evidence suggests that Treponema pailidum escapes humoral immune defence, although the host produces antibodies early in the infection. Heterologous antibodies are diagnostic evidence of syphilis and indicate polyclonal activation of B lym- phocytes. Conflicting results have been reported con- cerning cell mediated immunity status at different stages of the disease. Badanoiu et al,' Janot et al,2 and Schell and Musher,3 are among the workers who have produced evidence of good in vitro cellular immune response against Tpallidum. Other studies of delayed type hypersensitivity and inhibited leucocyte migra- tion have indicated diminished cell mediated immun- ity to Tpallidum in early syphilis.' Gschnait et al have claimed that hitherto undetermined serum factors possibly block the immune system in the blood of patients with syphilis.8 In the study reported here we examined the identity of mononuclear cells and subsets that made up the lesional infiltrate at different stages of syphilis, both before and after penicillin administration, and speculated on the local pathogenetic mechanisms during the course of the disease. Address for reprints: Dr A D Tosca, Department of Dermatology, "Andreas Sygros" Hospital, 5 Dragumi Street, Kesariani 161 21, Athens, Greece Accepted for publication 4 April 1988 Patients, materials, and methods We studied skin biopsy specimens from 40 patients with early syphilitic lesions who were divided into four groups. Group 1 consisted of 11 with primary syphilis who were seronegative but dark field positive. Group 2a consisted of six similar patients 24 hours after they had received an intramuscular injection of 2.4 MIU benzathine penicillin. Group 2b consisted of four similar patients after intramuscular administration of a total of 3.6 MIU. Group 3 consisted of 11 patients with secondary syphilis (six with macular, and five with papular lesions) who were seropositive in the quantitative Venereal Disease Research Laboratory (VDRL), Wassermann, and fluorescent treponemal antibody absorption (FTA-ABS) (IgG and IgM) tests and the Tpallidum haemagglutination assay (TPHA). Group 4a consisted of five patients similar to those in group 3 24 hours after receiving 2-4 MIU benzathine penicillin. Group 4b consisted of three similar patients after receiving 3.6 MIU. Both 2b and 4b groups were given 1-2 MIU one week after the 2-4 MIU penicillin. Patients with syphilis and positive for antibodies to HIV were excluded from the study. The methods used were the indirect immuno- peroxidase and the alkaline phosphatase anti-alkaline phosphatase (APAAP) techniques with monoclonal antibodies on 5p m cryostat sections fixed with acetone. The following antibodies were used: OKT3 289 on October 22, 2021 by guest. Protected by copyright. http://sti.bmj.com/ Genitourin Med: first published as 10.1136/sti.64.5.289 on 1 October 1988. Downloaded from
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Genitourin Med 1988;64:289-93
Infiltrate of syphilitic lesions before and aftertreatmentA TOSCA, J LEHOU, M HATJIVASILIOU, A VARELZIDIS, JD STRATIGOS
From the Department ofDermatology, "Andreas Sygros" Hospital, School ofMedicine, National University ofAthens, Athens, Greece
sumMARY An immunohistological study of skin biopsy specimens from patients with early syphiliswas undertaken before and after treatment (one day after intramuscular administration of 2.4 MIUbenzathine penicillin and eight days later, after a total administration of 3.6 MIU.
In chancres from seronegative patients treatment with 3.6 MIU usually resulted in fewerimmunocompetent cells in the infiltrate. In lesions of secondary syphilis treatment with 2.4 MIUbenzathine penicillin produced a significant decrease in immunocompetent cells. After treatment with3.6 MIU there was no further decrease. It was worth noticing that even eight to nine days after theinitial pretreatment biopsy, when 3.6 MIU had been administered, the overall lymphohistiocyticinfiltrate was not substantially diminished. Significantly more suppressor (T8+) cells were found inlesions of primary syphilis than of secondary syphilis, and they showed remarkable exocytosis.Activated local T8+ cells may release immunosuppressive lymphokines.
Abundant evidence suggests that Treponema pailidumescapes humoral immune defence, although the hostproduces antibodies early in the infection.Heterologous antibodies are diagnostic evidence ofsyphilis and indicate polyclonal activation of B lym-phocytes. Conflicting results have been reported con-cerning cell mediated immunity status at differentstages of the disease. Badanoiu et al,' Janot et al,2 andSchell and Musher,3 are among the workers who haveproduced evidence of good in vitro cellular immuneresponse against Tpallidum. Other studies of delayedtype hypersensitivity and inhibited leucocyte migra-tion have indicated diminished cell mediated immun-ity to Tpallidum in early syphilis.' Gschnait et al haveclaimed that hitherto undetermined serum factorspossibly block the immune system in the blood ofpatients with syphilis.8
In the study reported here we examined the identityof mononuclear cells and subsets that made up thelesional infiltrate at different stages of syphilis, bothbefore and after penicillin administration, andspeculated on the local pathogenetic mechanismsduring the course of the disease.
Address for reprints: Dr A D Tosca, Department of Dermatology,"Andreas Sygros" Hospital, 5 Dragumi Street, Kesariani 161 21,Athens, Greece
Accepted for publication 4 April 1988
Patients, materials, and methods
We studied skin biopsy specimens from 40 patientswith early syphilitic lesions who were divided into fourgroups. Group 1 consisted of 11 with primary syphiliswho were seronegative but dark field positive. Group2a consisted of six similar patients 24 hours after theyhad received an intramuscular injection of 2.4 MIUbenzathine penicillin. Group 2b consisted of foursimilar patients after intramuscular administration ofa total of 3.6 MIU. Group 3 consisted of 11 patientswith secondary syphilis (six with macular, and fivewith papular lesions) who were seropositive in thequantitative Venereal Disease Research Laboratory(VDRL), Wassermann, and fluorescent treponemalantibody absorption (FTA-ABS) (IgG and IgM) testsand the Tpallidum haemagglutination assay (TPHA).Group 4a consisted of five patients similar to those ingroup 3 24 hours after receiving 2-4 MIU benzathinepenicillin. Group 4b consisted of three similar patientsafter receiving 3.6 MIU. Both 2b and 4b groups weregiven 1-2 MIU one week after the 2-4 MIU penicillin.Patients with syphilis and positive for antibodies toHIV were excluded from the study.The methods used were the indirect immuno-
peroxidase and the alkaline phosphatase anti-alkalinephosphatase (APAAP) techniques with monoclonalantibodies on 5p m cryostat sections fixed withacetone. The following antibodies were used: OKT3
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290(directed towards the T3 complex ofmature peripheralT lymphocytes), OKT4/DAKO-T4 (predominantlyfor helper (inducer) cells), OKT8 (for suppressor(cytotoxic) T lymphocytes), Na,34 (for dendritic(mainly Langerhans') cells and activated T cells), Bl(for B cells), and OKM 1 (for monocytes/macro-phages). OKT3, OKT4, OKT8, and OKM 1 were
purchased from Ortho Pharmaceutical (Raritan, NewYork, USA), DAKO-T4 from Dakopatts (Denmark),Na,34 from Seralab (Sussex, England), and Bl fromCoulter Electronics (Luton, England). The details ofthe indirect immunoperoxidase procedure have beendescribed previously.9 APAAP was undertaken accor-ding to the protocol of Schaumburg-Lever.'0 It was
considered to be more sensitive and was used to checkthe results obtained with the indirect immunoperox-idase method. The results were always similar. OKT4and DAKO-T4 gave similar results in the 12 specimensused. The percentage of cells staining positively withthe "helper" antibodies was almost equal to that withperipheral T cell antibodies.9`'The cell subpopulations in the dermal infiltrate were
given as percentages of lymphoid cells. Dendritic cellsin the epidermis (Na,34+) were counted using an
ocular micrometer (Zeiss, WKPL, 10 x ) on a lightmicroscope ( x 400 magnification) and were expressedas cells/mm2 epidermal cell surface.The histological results were evaluated as intense
disperse perivascular infiltrate, perivascular and in thedermal papillae, perivascular infiltrate only, or
negative.Cellular components were classified as (+ + +) if
the cell type predominated in the infiltrate, (+ +) ifthere were many cells, ( + ) if they could be observed inadequate numbers, (±) if they were rarely observed,and (-) if there were none.
Statistical analyses were undertaken using theWilcoxon rank sum test.
Results
From the histological study (table 1) vertical correla-
Fig 1 Exocytosis of T8+ cells into epidermis ofpapulefrompatient with untreatedprimary syphilis. (Alkalinephosphatase anti-alkaline phosphatase.)
tion between groups did not show significant differen-ces, except that significantly (p < 0.05) fewer plasmacells were found in group 2b than in group 1. Moreplasma cells were found in group 3, although notsignificantly more than in group 1.The lymphohistiocytic infiltrate was mainly
Table 1 Histologicalfindings (figures show mean (SD) positivity graded on an arbitrary scalefrom 0 to 4)
MacrophagesGroup No Infiltrate Lymphocytes (histiocytes) Plasma cells Polymorphs Eosinophils
Groups 1 and 2 = patients with primary syphilis, group 2a 24 hours after receiving 2-4 MIU benzathine penicillin, group 2b after receiving3-6 MIU; groups 3 and 4 = patients with secondary syphilis, group 4a 24 hours after receiving 2-4 MIU, group 4b after receiving 3-6 MIU.The presence of polymorphs and eosinophils was not correlated.*Two additional group 3 specimens included in histological study only.
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See footnote of table I for descriptions of groups.Results not available from one group I patient for BI + and Na,34+.
subepidermal and perivascular, and it persisted evenafter treatment with 3.6 MIU penicillin.The immunohistochemical study showed a charac-
teristic exocytosis ofT8+ cells (fig 1) that was promin-ent mainly before treatment. Minor exocytosis ofT3 +,T4+, and OKM1 + cells could be observed in somecases. Dermal Na,34+ cells were predominantly lym-phoid, although dendritic cells could also be observedin the upper dermis.
Table 2 shows semiquantitative analysis of the dataobtained. Differences between groups 1 and 2a weresignificant regarding T4+ cells (p < 005) and showeda trend towards significance for T3 + andOKM 1 + cells
and Na,34+ epidermal dendritic cells. Group 2b hadsignificantly fewer T3+ and T4+ (p < 005), T8+ (p -
005), and Bl+ (p < 0-01) cells than group 1, andNa,34+ dermal cells showed a trend towards decreasewithout reaching significance. The decrease in T8 + andB1+ cells in group 2b was significant even comparedwith group 2a (p 0.05 and p < 0-01, respectively).Comparison between (untreated) groups 1 and 3
showed significantly more T8+ cells (p < 0-01) (fig 2)and Bl+ cells (p < 005) in group 3 and a trendtowards more dendritric epidermal cells.
Significantly fewer T3+ (p - 005), T8+ (p < 005),and Na,34+ dermal cells (p < 0-01) were seen in group
4a than group 3 patients, as well as a trend towardssignificantly fewer T4+ andOKM 1 + cells. Correlationwith group 4b was not undertaken because it con-tained so few patients. The differences between 4a and4b, however, seemed to be negligible.When comparing the epidermal denditric cells in
groups 1 and 3 with those in normal skin specimens,'2significantly fewer were found (p < 0.05) in skinspecimens from group 1 than in normal skin.When comparing the local T4V and T8+ dermal
infiltrates ofgroups 1 and 3 with those ofpatients withlichen planus,'3 significantly lower values were foundin group 1 (T4V cells, p < 0.01; T8+ cells, p < 0.01)and in group 3 (T4V cells, p < 0-01; T8+ cells, p0.05).
Discussion
This study showed significantly fewer epidermal den-dritic cells in group 1 skin specimens than in normalskin, which is compatible with the hypothesis thatreduced numbers of Langerhans' cells in the skinmight enhance local multiplication of treponemes.'4Significantly smaller percentages ofT4+ and T8+ cellsin the inflammatory infiltrate of group 1 and 3specimens than in that of patients with lichen planus'3favours the hypothesis of somehow decreased local
cellular immune events in the inflammatory infiltrateof lesions of early syphilis. Treating patients withprimary syphilis with 3-6 MIU penicillin usuallyreduced immunocompetent cells in the infiltrate.Treatment of patients with secondary syphilis showedan earlier response, producing an appreciable decreaseafter 2.4 MIU benzathine penicillin. Therafter,however, the rate of decrease appeared to be slow.
Correlating the histological with the immunohisto-chemical findings we can seen that, although thepercentages of B and T lymphoid cell subpopulationsof the infiltrate changed after treatment and weredecreasing at their own particular rates, even after atotal of 3-6 MIU penicillin had been administeredeight to nine days after the initial pretreatment biopsy,the overall lymphohistiocytic infiltrate was not sub-stantially reduced.T cells and subsets have been considered to be
locally important in the early stages of syphilis, asBjerke et al postulated for the interaction of host andparasite at that stage of the infectious process,'" desp-ite the fact that systemically their function might beimpaired by serum factors (such as spirochaeteproducts, immune complexes or host immunosuppres-sive substances).Our results showed a delayed influence of treatment
on locally distributed immunocompetent cells. This is
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Infiltrate ofsyphilitic lesions before and after treatmentconsistent with the results of Friedman and Turk whofound that people undergoing treatment with drugsshowed pronounced lymphocytic reactivity to the Tpallidum antigen."
In syphilis, Tpallidum stimulates local cell mediatedimmunity that leads to its elimination.3 Macrophageshelped by Langerhans' cells undertake phagocytosisand process the antigen, which has specific biologicalproperties. Moreover the spirochaete is protected by amucoid envelope that prevents its being engulfed byphagocytic cells or those presenting antigen. Jensenand From have suggested that macrophages areinhibited by infected T cells, which render C3f recep-tors unavailable for processing antibody complementopsonised treponemes.`More Na,34+ epidermal dendritic cells (fig 3) were
found in patients with secondary than primarysyphilis. This was consistent with the findings ofMittag and Klingmiiller, who also found severalLangerhans' cells in one patient with granulomatoussyphilis (a transitional stage between early and latesyphilis).'4 They considered that the interaction ofLangerhans' cells with intraepidermal T pallidummight be important for the epidermotropic infiltra-tion.
Decreased numbers of circulating suppressor cellshave been reported in secondary syphilis,'8 which maypartly account for the increased amount ofheterologous antibodies during this stage. The relativeincrease in locally distributed T8+ cells at this stage (fig2) might be related to the decrease in blood, because ofthe peripheral distribution of lymphocytes in selectivetissues.'9 Increased T8+ cells possibly releaseimmunosuppressive lymphokines20 locally and intothe blood stream, giving the invading micro-organisman advantage for long term survival.8 This, in conjunc-tion with local antibody releasing cells (B cells andplasma cells) might explain the serological findings inadvanced primary or secondary syphilis.
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